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Supporting Information
Kinoshita et al. 10.1073/pnas.1418896111
SI Materials and Methods
TRAP and RNA-Seq Analyses. TRAP was performed as described
in Heiman et al. (1). Briefly, hippocampal tissue from Gprin3–
EFGP–L10 mice (n = 6) was manually homogenized and centrifuged at 2,000 × g for 10 min at 4 °C, and supernatants were incubated with Nonidet P-40 for 5 min on ice before a 15-min spin
at 20,000 × g at 4 °C. Supernatant was applied to Streptavidin
MyOne T1 Dynabeads (Invitrogen no. 65601) that had been
incubated with biotinylated protein-L (Pierce no. 29997) and
α-EGFP antibodies [19C8 and 19F7 custom from the Memorial
Sloan Kettering Cancer Center core facility (1)] overnight at 4 °C.
After immunoprecipitation, unbound fractions were saved, and
beads were washed five times with 0.35 M KCl wash buffer before
beads were resuspended in lysis buffer (Stratagene Absolutely
RNA Nanoprep Kit no. 400753). RNA purification was performed per the manufacturer’s instructions; quantification and
RNA integrity were determined by using a Bioanalyzer (Agilent), and only samples with RNA Integrity Numbers greater
than eight were used for RNA-Seq.
Samples were prepared for sequencing by the Rockefeller
University Genomics Core Facility using the TrueSeq RNA
Sample Preparation Kit v2 (Illumina) and barcoded to allow
samples to be multiplexed within a flow cell lane. Barcoded cDNA
libraries were sequenced on an Illumina HiSeq 2000 in a single
lane to obtain 100-bp single-end reads at an approximate sequencing depth of 25–30 million reads per sample. Raw reads
were aligned to mouse genome (mm9) by using TopHat2 (2).
Reads were filtered on quality-control metrics (reads with QC <
30 were discarded), and differential expression analysis was
conducted by using Genespring (Agilent) to obtain Z scores and
fold change values for individual genes (Fig. S2). Differences in
integrated read density were visualized against the mouse genome by using Genespring (or IGV; Broad) (Figs. S3 and S4).
Antibodies and Immunofluorescence Microscopy. Antibodies purchased from Santa Cruz Biotechnology were as follows: NUP62
rabbit polyclonal (sc-25523), Ankyrin G mouse monoclonal (sc12719), MAP2 rabbit polyclonal (sc-20172), mouse monoclonal
antibody Tau46 (binds tau protein and MAP2; sc-32274), and
PYK2 rabbit polyclonal (sc-9019). Mouse monoclonal antibody to
NUP62 uncoupled or coupled to FITC was purchased from BD
Biosciences (611692 and 610497). Mouse monoclonal antibody to
nonphosphorylated (at Ser-199/202/205) tau protein was purchased
from Cedarlane (Tau-1; CLT9007). Mouse monoclonal antibody to
tau phosphorylated at Ser-202 (monoclonal CP13) was provided by
Peter Davies (Albert Einstein College of Medicine, Bronx, NY).
Mouse monoclonal antibody to NUP133 was purchased from
Abnova (1069-1155, H00055746-M01). Rabbit polyclonal antibodies to phospho-Y402 PYK2 and NUP98 were purchased from
Cell Signaling (3291S and 2598S, respectively). Monoclonal antibody to GAPDH was purchased from Millipore (MAB374).
Monoclonal anti-V5 epitope antibody was purchased from Thermo
Scientific (product no. MA1-81617).
Characterization and/or verification of the antibodies used in
this study was performed as follows. The NUP62 rabbit polyclonal, NUP62 monoclonal antibody, and NUP133 monoclonal
antibody have been characterized by Y.K. and D.S.K. in published
studies in which identification of Western blot bands for NUP62
and NUP133 was confirmed by siRNA knockdown analyses (3, 4).
In addition, the monoclonal NUP62 and NUP133 antibodies
were shown by immunofluorescence microscopy to produce a
pattern at the nuclear envelope that is consistent with nuclear
Kinoshita et al. www.pnas.org/cgi/content/short/1418896111
pores. Characterization and use of the following antibodies has
been reported in the following references: ankyrin G mouse
monoclonal antibody (5, 6), MAP2 rabbit polyclonal antibody (7,
8), Tau46 mouse monoclonal antibody (9), PYK2 rabbit polyclonal antibody (10, 11), Tau-1 mouse monoclonal antibody (12,
13), CP13 mouse monoclonal antibody (14, 15), rabbit polyclonal
phospho-Y402 PYK2 (16, 17), and mouse monoclonal antibody
to GAPDH (4, 18). In Western blot analyses, the NUP98 rabbit
polyclonal antibody bound a major band that migrated at 98 kDa
and two much lighter more rapidly migrating bands (Fig. S5). We
used only the 98-kDa band for quantification in nuclear extracts.
In further confirmation of specificity, the NUP98 antibody produced nuclear envelope rim and intranuclear filament patterns
in immunofluorescence analysis (Fig. S5), as described for other
NUP98 antibodies (19).
Polyclonal rabbit antibody to NUP62 phosphorylated at Y422
was developed in rabbits immunized with the synthetic peptide
KEQSGTIY*LQHADEE (* refers to phos) conjugated to KLH.
Antibodies were affinity purified with the phosphorylated peptide and were affinity depleted with nonphosphorylated peptide.
The antibodies were prepared and purified by GenScript. The
antibodies bound a dexamethasone-induced major band in S12
whole-cell extracts that comigrated at 62 kDa with NUP62 bands
identified by generic NUP62 antibodies (Fig. S5). A major band
migrating at the same position was observed in whole and cytosolic extracts from rat hippocampus (Fig. S5). Because of the
presence of additional bands in Western blot (which is typical for
phospho-tyrosine–directed antibodies), sequential immunoprecipitation/Western blot experiments were presented in this work
for the phospho-Y422 NUP62 antibody.
Cultured cells were fixed and processed for immunofluorescence microscopy as described (3). Brain sections were treated
with 0.5% Triton X-100 in HMK buffer (20 mM Hepes, pH 7.5,
1 mM MgCl2, 100 mM KCl) for 20 min. Sections were treated with
blocking solution [10% normal donkey serum and 1% BSA (Sigma)
in HMK buffer] for 30 min and then incubated with primary antibody for 18–24 h at 4 °C with shaking. After washing with three
changes of HMK, sections were incubated with secondary antibodies (Jackson ImmunoResearch) for 1.5 h in HMK supplemented with 1% BSA. Sections were washed in HMK four times
for 10 min each at room temperature with shaking, and then rinsed
once with water and mounted in VectaShield mounting medium
with DAPI (Vector). Secondary antibodies for immunofluorescence
microscopy were purchased from Jackson ImmunoResearch.
Cell and Tissue Fractionation, Immunoprecipitation, and Western Blot
Analyses. Cultured cells and resected hippocampi were extracted
into whole-cell, cytoplasmic, and nuclear fractions by the REAP
method as described (20). Aliquots of each fraction representing
the yield from equivalent amounts of starting material were supplemented with 6× SDS sample buffer to render them 1× [1× SDS
sample buffer: 62.5 mM Tris·HCl (pH 6.8), 10% glycerol, 2%
SDS, 5% beta-mercaptoethanol, 125 μg/mL bromophenol blue].
Dexamethasone was purchased from Sigma-Aldrich. Pervanadate
was prepared fresh from a solution of 200 mM sodium orthovanadate (Sigma-Aldrich) by adding hydrogen peroxide to a final
concentration of 0.18% and incubating for 15 min. The FAK/PYK2
inhibitor PF562271 was purchased from Selleckchem. For immunoprecipitation, cultured cells were lysed in radioimmunoprecipitation assay (RIPA) buffer [20 mM Tris·HCl (pH 7.5),
150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Nonidet
P-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate,
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1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin] and clarified by centrifugation. Lysates were preadsorbed
with Protein G coupled to agarose beads (Cell Signaling), clarified by centrifugation, and then incubated with primary antibody
overnight (4 °C). Antibody and bound proteins were collected by
incubation with Protein G coupled to agarose, and the beads were
washed and pelleted by centrifugation four times with RIPA buffer.
Bound proteins were eluted by incubation in 1× SDS sample buffer
at 80 °C for 10 min. Whole-cell lysates for cultured cells were prepared by washing cells once with PBS followed by resuspension in
1× SDS sample buffer.
Methods for SDS/PAGE and immunoblot transfers of proteins
to nitrocellulose membranes are detailed in the manual for the
Amersham ECL Plus Western blotting system (GE Healthcare).
Membranes were incubated with primary and secondary antibodies by using the ECL Plus systems as described (3). Membranes were exposed to X-ray films and band densities were
determined from digital scans by using Image J (Version 1.45i;
imagej.nih.gov/ij). Student’s t test analyses, ANOVA, and
posttests were performed by using online calculators.
siRNA Knockdown and Rescue. After plating, cells were allowed to
grow for 1–7 d before transfection. Cells were transfected siRNA
or siRNA plus V5–NUP62 expression construct (3) with Lipofectamine RNAiMAX (Invitrogen) as per the manufacturer’s
recommendations for 72 h. For most experiments, Santa Cruz
Biotechnology hNUP62 siRNA sc-36107 [sc-36107A (sense:
CUGCAGCAGAUCUGCAAGAtt) targets rat NUP62 sequence]
was used. This siRNA contains at least three mismatches with any
other rat mRNA in the Refseq dataset. Dendritic retraction was
confirmed by using Santa Cruz siRNA sc-36108 [sc-36108A (sense:
CUGGAAAGCUUGAUCAACAtt) would target rat NUP62, but
is less potent than sc-30107A because of a single nucleotide mismatch]. The sc-36107 mix contains equal portions of sc-36107A
and two other siRNAs that do not present >16 nucleotide matches
to any Refseq rat RNAs. In experiments where sc36107 was
transfected at 150 nM to effect 50 nM sc-36107A, 150 nM control
siRNA was used. The reported concentrations of NUP62 siRNA
should be adjusted per batch to generate equivalent dose-related
effects. Control siRNA-A (sc-37007) and control siRNA-B (sc44230) were purchased from Santa Cruz Biotechnology.
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Fig. S1. Hippocampal expression of eGFP–L10a from the Gprin3 promoter. Frozen sections of brain from a transgenic mouse strain bearing a BAC with the
Gprin3 upstream region driving expression of eGFP–L10a were processed for and viewed by eGFP fluorescence microscopy. Expression of eGFP–L10a is concentrated in the CA3 subfield of the hippocampus.
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Fig. S2. RNA-Seq comparative analysis of TRAP RNAs from CA3 of control and chronically restraint-stressed Gprin3 bacTRAP mice. Fold changes in translated
transcripts that were statistically inferred to be significant are shown as a signal ratio of control to chronically stressed. As shown in Fig. S3, reads for the
Nup62–Il4i1 gene fall entirely within the Nup62 coding region. n.s., not significant.
Fig. S3. Chronic restraint stress significantly decreases Nup62 mRNA translation in murine CA3 neurons. RNA-Seq analysis of TRAP mRNAs revealed decreased
Nup62 mRNA. Base and alignment quality or Z test were performed by using Genespring (Agilent Technologies). Control and chronic restraint stress (CRS)derived RNAs were sequenced to 25 million 100-bp end reads.
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Fig. S4. Distribution of Nup62–Il4i1 RNA-Seq reads between Nup62 and Il4i1 coding regions by using TRAP isolated RNAs from CA3 of control and chronic
restraint stressed (CRS) Gprin3 bacTRAP mice. Alignment of the RNA-Seq data was performed with the Integrated Genomics Viewer (IGV).
Fig. S5. (A) Western blot analysis (WB) of hippocampal nuclear extract with NUP98 antibody. Arrowhead, position of band used for quantifications. (B)
Immunofluorescence microscopy of an ovarian carcinoma cell with antibodies to NUP98 and NUP62. Images are projections of 3D deconvolved z-stacks. (Bar:
5 μm.) (C) Phospho-Y422 NUP62 antibody WB of whole-cell lysates of untreated S12 cells (Con) and S12 cells treated with dexamethasone (Dex) or F562271
inhibitor (In.) and dexamethasone. Arrowhead, position of NUP62. (D) Phospho-Y422 NUP62 antibody WB of cytosol from control (Con) and chronically stressed
(Str) rat hippocampus. Arrowhead, position of NUP62. (E) S12 cells were treated with (a) nothing, (b) dexamethasone for 1.5 h, or (c) dexamethasone and
PF562271 inhibitor for 1.5 h. WB with phospho-Y422 NUP62 antibody (Ab6) was performed on NUP62 monoclonal antibody immunoprecipitates; membranes
were then stripped and blotted with rabbit antibody to total NUP62. Inhibitor was not fully effective in this experiment because it was added simultaneously
with activator. (F) S12 cells were treated as in E, but immunoprecipitates were generated with unrelated mouse IgG2b antibody.
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Fig. S6. Western blot analyses of Pyk2 (Total Pyk22) and Pyk2 phosphorylated at Y402 (Phospho-Y402 Pyk2) in whole-cell, nuclear, and cytoplasmic extracts
from control and chronically stressed rats. Densities of Western blot signals were compiled and compared. Only P values <0.05 are shown.
Fig. S7. Immunofluorescence microscopy using Nup62 (green) or Phos-Y402 Pyk2 (P-Pyk2; red) antibodies. DNA is stained with DAPI (blue). Shown is an optical
section resolved by apotome optics and deconvolution in the CA3 region of hippocampus from a chronically stressed mouse. P-Pyk2 signal is observed in the
cytoplasm adjacent to the nuclear envelope as well as adjacent to the nuclear envelope within the nucleus. Microglial cells (smaller nuclei) display much less
Nup62 signal and a different chromatin distribution pattern. (Bar: 5 μm.)
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Fig. S8. Low-power montage of immunofluorescence images of hippocampus from control (Control) or chronically stressed (Stress) mice. Sections were labeled with Nup62 (green) or Phos-Y402 Pyk2 (P-Pyk2; red) antibodies. Nuclei are stained with DAPI (blue). (Bar: 200 μm. The 3D thermal surface plots of intensity (thermal color scale) reveal in stress images that enhancement of somal P-Pyk2 signal is most apparent in CA3.
Fig. S9. Forced expression of V5 epitope-tagged NUP62 in neurons transfected with NUP62 siRNA results in extended process formation. Primary rat E18
hippocampal neurons at seven days in vitro (DIV) were cotransfected with 33 nM NUP62 siRNA and 1 μg of an expression plasmid for V5 epitope-tagged NUP62
(3). At 12 DIV, cells were fixed and labeled with V5 epitope tag (green) and MAP2B (red) antibodies. (Bar: 25 μm.)
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Fig. S10. Forced expression of V5 epitope-tagged NUP62 in neurons transfected with NUP62 siRNA results in extended process formation. Primary rat E18
hippocampal neurons at seven DIV were cotransfected with 33 nM Control or NUP62 siRNA and 1 μg of an expression plasmid for V5 epitope-tagged NUP62 (3).
At 12 DIV, cells were fixed and labeled with V5 epitope tag (green) and MAP2B (red) antibodies. (A) Total length of processes was quantified for Control siRNA
and NUP62 siRNA transfected cells, and NUP62 s RNA transfected cells expressing V5–NUP62. Shown are mean process lengths per cell and SD. (B) ANOVA table
for three groups, showing sum of squares (SS), degrees of freedom (df), mean squares (MS), and F and P values given the number of subjects per group. (C)
Post-test comparisons using Bonferroni corrections, showing significance and t values. (D) Post-test comparisons showing 95% confidence intervals.
Fig. S11. Reduced Nup62 content impedes dendritic development in differentiating rat E18 hippocampal neurons in culture. (Upper) Rat E18 hippocampal
neurons at one (day 1) or three (day 3) DIV were transfected with control or 50 nM NUP62 siRNA. At 10 DIV, cells were fixed and processed for immunofluorescence microscopy with antibodies to ankyrin-G (Ank3; green pseudocolor) and Map2 (red pseudocolor). Dendrites appear orange/yellow and axons
appear green. Nuclei were stained with DAPI. (Bar: 100 μm.)
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