EMBO/soda-nel huggingface dataset:
Unfolded Protein Response-Induced ERdj3 Secretion Links ER Stress to Extracellular Proteostasis (2015)
Figure 2

Figure 2. ERdj3 is efficiently secreted from cells following stress-independent activation of the UPR-associated transcription factor ATF6.

Immunoblot in lysates and media collected from HEK293DAX cells following 16 h of activation of XBP1s [by 1 g/mL dox] and/or ATF6 [by 10 M TMP], as indicated (Shoulders et al, 2013). Immunoblots of the ATF6 target protein BiP (reactive with the KDEL antibody) and the XBP1s target Sec24D confirm the small molecule activation of these transcription factors.
Representative autoradiogram of [35S]-labeled ERdj3 immunopurified from lysates and media collected from HEK293DAX cells following preactivation of XBP1s [by 1 g/mL dox] and/or ATF6 [by 10 M TMP] (Shoulders et al, 2013). The experimental protocol is shown above.
Quantification of relative amounts of [35S]-labeled ERdj3 in media at 8 h collected from cells treated as shown in Figure 2B (n=3). The media [35S]-labeled ERdj3 was measured by densitometry and is normalized to the amount of media [35S]-labeled ERdj3 at 8 h from vehicle-treated cells. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01; *** indicates p-value < 0.001 as compared to the vehicle-treated condition.
Quantification of the fraction ERdj3 secreted from autoradiograms as shown in Figure 2B. The fraction of ERdj3 secreted was calculated by normalizing the recovered [35S]-labeled ERdj3 signal in the media at t = 4 or 8 h to the total amount of [35S]-labeled ERdj3 signal in the media and lysates at t = 0 h. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01 as compared to the vehicle-treated condition.
Quantification of the fraction ERdj3 remaining from autoradiograms as shown in Figure 2B. The fraction of ERdj3 remaining was calculated by normalizing the sum of the recovered [35S]-labeled ERdj3 signal in the media and in the lysates at t = 4 or 8 h to the total amount of [35S]-labeled ERdj3 signal in the media and lysates at t = 0 h. Error bars represent standard error from biological replicates (n = 3). * indicates p-value < 0.05; ** indicates p-value < 0.01 as compared to the vehicle-treated condition.
Unfolded Protein Response-Induced ERdj3 Secretion Links ER Stress to Extracellular Proteostasis (2015)
Figure 3

Figure 3. Secreted ERdj3 inhibits extracellular protein aggregation

Immunoblot of conditioned media collected from HEK293T cells transiently transfected with ERdj3WT or ERdj3H53Q and incubated with an affinity resin consisting of native (N) or guanidinium hydrochloride (GdnHCl)-denatured (D) RNAse A covalently coupled to sepharose resin. Resin was incubated with media for 2 h at ambient temperature, followed by washing and elution in Laemmli's reducing buffer.

Plot showing the time-dependent increase in ThioT fluorescence (ex:440 nm, em:485 nm) of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation in the presence or absence (black) of recombinant ERdj3 (RERdj3). The RERdj3 concentrations and molar ratios of RERdj3 to A1-40 are as follows: Violet: 1.5 nM, 1:6561; light blue: 4.6 nM, 1:2187; dark blue: 14 nM, 1:729; dark green: 41 nM, 1:243; light green: 120 nM, 1:81; yellow: 370 nM, 1:27; orange: 1.1 M, 1:9; red: 3.3 M, 1:3, or maroon: 10 M, 1:1. All traces represent the average of three replicates.

Plot showing the time-dependent increase in ThioT fluorescence of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation in the presence or absence of BSA (14 µg/mL) or RERdj3 (14 µg/mL; 370 nM, RERdj3: A=1:27). All traces represent the average of three replicates.

Immunoblot of A1-40 incubated (10 µM, 37 OC, pH 7.2) with regular agitation for 90 h in the presence or absence of RERdj3 (14 µg/mL; 370 nM, RERdj3: A1-40 =1:27) or BSA (14 µg/mL). The reaction solution in the well was collected (Nonadhered) and separated into soluble and insoluble fractions by centrifugation. These samples, as well as the washed wells of the plate (Adhered), were denatured in 8 M GdnHCl with sonication and analyzed by SDS-PAGE/immunoblotting.
Unfolded Protein Response-Induced ERdj3 Secretion Links ER Stress to Extracellular Proteostasis (2015)
Figure 4

Figure 4. Secreted ERdj3 attenuates vacuole formation induced by toxic prion protein in mammalian cells.

Schematic illustrating the treatment of PK1 cells with ERdj3-conditioned media. HEK293T-Rex (Control) and HEK293T-Rex cells stably overexpressing tet-inducible ERdj3WT on poly-D-lysine coated plates were treated with dox (1 g/mL) overnight to induce ERdj3WT expression. The media was replaced with fresh OptiMEM I + 5% FBS, which was conditioned for 30 h, sterile-filtered, concentrated 5 X against a 3 kD MWCO membrane, and diluted 1:1.5 with fresh OptiMEM I + 5% FBS. PK1 cells were plated in OptiMEM I + 5% FBS, and after 24 h were treated with conditioned media and TPrP (600 ng/mL) for 3 d.
Representative images under 136X magnification of PK1 cells incubated with control conditioned media collected from HEK293T-Rex or conditioned media prepared from HEK293T-Rex cells overexpressing ERdj3WT in the presence or absence of TPrP (600 ng/mL), as described in Figure 4A. Arrows indicate representative cells containing vacuoles. Insets show 544X magnification of a typical region of the image.
Quantification of vacuole formation from images as shown in Figure 4B. Equal area images comprising ~1200 cells per well were counted for each sample. Counting of cells and of cells with identifiable vacuoles was done using the Fiji image processing package (Schindelin et al, 2012). *** indicates a p-value < 0.001 (n = 3).
Unfolded Protein Response-Induced ERdj3 Secretion Links ER Stress to Extracellular Proteostasis (2015)
Figure 5

Figure 5. ERdj3 is co-secreted with destabilized proteins through the secretory pathway.

Schematic describing the co-incubation and co-expression experiments used to demonstrate ERdj3 co-secretion with destabilized TTR. For co-incubation, HEK293T cells overexpressing flag-tagged TTR variants were seeded with an equal number of cells overexpressing ERdj3. For co-expression experiments, HEK293T cells are transfected with both flag-tagged TTR and ERdj3. TTR was immunoisolated with M2 anti-FLAG beads from media conditioned overnight on the cells, eluted and separated by SDS-PAGE for immunoblotting.

Immunoblot of M2 anti-FLAG immunopurifications from the conditioned media collected from HEK293T cells overexpressing FTTTRWT, FTTTRA25T, and/or ERdj3WT either as a co-incubation or co-expression experiment as shown in Figure 5A. Beads were washed in RIPA buffer prior to elution. Media inputs (1:400) are shown as a control.

Immunoblot of M2 anti-FLAG immunopurifications from media collected from HEK293T cells overexpressing ERdj3WT and FTTTR variants as indicated. Media inputs (1:400) are shown as a control.

Immunoblot of M2 anti-FLAG immunopurifications from conditioned media of HEK293T cells overexpressing FTTTRA25T and ERdj3WT in the presence of the indicated concentrations of Tafamidis, a kinetic stabilizer of TTR tetramers. Media inputs (1:400) are shown as a control.

Immunoblot of 6E10 anti-APP immunopurifications from the conditioned media of CHO cells or CHO-derived APP751-expressing (7WD10) cells overexpressing ERdj3WT as indicated. Cells were replated together as indicated 24 h after transfection at equal stoichiometry, and media conditioned for 36 h. In this case, ERdj3 transfected CHO cells replated with mock-transfected 7WD10 cells serves as the co-incubation condition, while mock-transfected CHO cells replated with ERdj3-transfected 7WD10 cells comprise the co-expression condition. Media inputs (1:150) are shown as a control.
Unfolded Protein Response-Induced ERdj3 Secretion Links ER Stress to Extracellular Proteostasis (2015)
Figure 6

Figure 6. ERdj3-client protein complexes are co-secreted when BiP activity is limiting.

Representative immunoblot of M2 anti-FLAG immunopurification of media conditioned for 24 h on HEK293DAX cells co-overexpressing FTTTRA25T and ERdj3WT or ERdj3H53Q. Media contained vehicle or trimethoprim (TMP, 10 M, 16 h; activates ATF6), as indicated. Media inputs (1:400) are shown as a control. Lysates from these cells are also shown to confirm the increase of the ATF6 target proteins BiP and GRP94 in cells following TMP-dependent activation of ATF6.

Bar graph depicting quantification of the change in the amount of ERdj3 co-immunoprecipitating with TTRA25T following activation of ATF6, as in Figure 6A. The ERdj3/TTR ratio is normalized to the ratio in the absence of ATF6 activation. *indicates a p-value < 0.05; n = 3.

Representative immunoblot of M2 anti-FLAG immunopurification of media conditioned for 24 h on HEK293T cells co-overexpressing FTTTRA25T, ERdj3WT, ERdj3H53Q, and/or BiP, as indicated. Media inputs (1:400) are shown as a control. Lysates from these cells are also shown to confirm the increase of BiP afforded by overexpression.

Quantification of immunoblots of M2 anti-FLAG immunopurification of media collected from cells co-overexpressing either ERdj3WT or ERdj3H53Q, FTTTRA25T, and BiP, as in Figure 6C. The ERdj3/TTR ratio is normalized to the ratio in the absence of BiP overexpression. **indicates a p-value < 0.01; n = 5.

Immunoblot of 6E10 anti-APP immunopurifications from the conditioned media of CHO cells or CHO-derived APP751-expressing (7WD10) cells overexpressing ERdj3WT, ERdj3H53Q, and BiP as indicated. Media inputs (1:100) are shown as a control. Lysates from these cells are also shown to confirm the increase of BiP afforded by overexpression.
Translation is required for miRNA-dependent decay of endogenous transcripts. (2020)
Figure 1

Figure 1- Steady-state abundance of lncRNAs is not directly affected by miRNA loss

(A) Percentage of mRNAs (n=6701, red) and predominantly cytosolic (n=57, blue) and nuclear lncRNAs (n=175, grey) with experimental evidence of binding by ribosomes (Translation Efficiency>0) in mESCs.

(B) Density of HEAP-AGO2 peaks across cytosolic lncRNAs (n=62, blue) and the 3'UTR regions of mRNAs (n=8798, red) with experimental evidence for AGO2 binding in mESCs (>0 AGO2 peaks). Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution.

(C, D) Small RNA and Poly(A)-selected RNA sequencing based estimates of the fold difference (y-axis) in (C) miRNA and (D) mRNA expression, respectively, relative to day 0, during a 12 days' time-course (x-axis) following treatment of DTCM23/49XY mESCs with 4-OHT and consequent loss of DICER function. Points represent the average miRNA or mRNA expression and error bars the standard deviation based on three independent biological replicates.

(E) Cumulative distribution plot of the fold-difference in expression after 8 days of 4-OHT treatment for mRNAs, relative to day 0 of treatment (tpm≥1) with (n=6034) and without (n=7887) evidence of AGO2-binding by HEAP (Li et al., 2020).

(F) Distribution of the relative fold-change after 8 days of 4-OHT treatment in steady-state abundance, relative to day 0 of treatment, for mESC expressed (tpm≥1) mRNAs (n=19306, red), cytosolic (n=445, blue) and nuclear (n=529, grey) lncRNAs (two-tailed Mann-Whitney U test, mRNAs vs cytosolic lncRNAs p-value<2X10-16 and cytosolic vs nuclear lncRNAs p=0.875). Statistics: NS-p-value>0.05 and ***-p-value<0.001. Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution. two-tailed Mann-Whitney U test.
Inflammation leads through PGE/EP3 signaling to HDAC5/MEF2-dependent transcription in cardiac myocytes (2018)
Figure 1

Figure 1 - Screening the effect of different GPCR-agonists on MEF2-activity.

A Neonatal rat ventricular myocytes (NRVMs) were infected with the 3xMEF2-Luc reporter, serum starved for 20 hours and stimulated for 24 hours with different agonists: 100 nM endothelin-1 (ET1), 1 μM sphingosine-1-phosphate (S1P), 10 μM lisophosphatidic acid (LPA), 1 μM WIN55,212-2 (WIN55, cannabinoid receptor agonist), 1 μM isoproterenol (ISO, β-adrenergic receptor agonist), 100 nM angiotensin II (AngII), 10 μM prostaglandin E1 (PGE1), 10 μM prostaglandin E2 (PGE2) or 100 nM prostanoid F receptor agonist fluprostenol (Flupro), 100 nM treprostinil (Trepro, prostacyclin receptor agonist).

B PGE2 induces the expression of MEF2 target genes. NRVMs were serum-starved for 24 hours, then were stimulated with 1 μM PGE2 for 2 hours and mRNA levels of the indicated genes were determined.

C Correlation between MEF2 activation and NRVM hypertrophy. MEF2 activity was measured as in (A) upon increasing concentration of PGE2. Cell size of NRVMs was determined after 48-hour stimulation with the indicated concentrations of PGE2. Left: representative images of α-actinin stained NRVMs stimulated with DMSO, 300 nM PGE2, or 1 nM ET1. Scale bar is 10 µm.

D, PGE2 activates MEF2 via EP3 receptor. NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were stimulated with DMSO or 1 μM PGE2 for 24 hours in the presence or absence of different EP receptor antagonists: AH6809 (10 μM, EP1- and EP2-antagonist) or 798106 (200 nM, EP3-antagonist) or L161,982 (2 μM, EP4-antagonist).

Data information: values are mean±s.e.m. In (A), the experiment was performed in triplicates, similar results were obtained in 3 different experiments. Student's two-tailed t-test, *,P<0.05 vs. control (ET1, P=0.0013; S1P, P=0.0144; LPA, P=0.026; WIN55, P=0.6205; ISO=0.0399; AngII, P=0.5812; PGE1, P=0.001; PGE2, P<0.0001; Flupro, P=0.0846; Trepro, P=0.1958); In (B), n=5, technical replicates, Student's two-tailed t-test, *,P<0.05 vs. control (Nur77, P=0.0148; Myomaxin, P=0.0417; Adamts1, P=0.0112; BNP, P=0.0065). In (C), NRVM cell size was determined from a minimum of 100 cells of 3 technical replicates upon different concentrations of PGE2, MEF2-activity was parallel assessed from 6 technical replicates. Correlation was statistically analyzed by calculating Pearson correlation coefficient (Pearson r=0.9626, P=0.0021). In (D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, AH6809, P=0.1092; L798106, P=0.0002; L161,982, P=0.5579). The exact n and P values can be also found in the Source Data excel file for Figure 1.
Inflammation leads through PGE/EP3 signaling to HDAC5/MEF2-dependent transcription in cardiac myocytes (2018)
Figure 2

Figure 2 - PGE2-mediated MEF2 activation is Gi/o-βγ dependent.

A Illustration of the target points of the used pharmacological and protein-based inhibitors.

B-D (B, D) NRVMs were infected with the 3xMEF2-Luciferase reporter and with recombinant adenoviruses encoding EGFP (AdGFP), RGS16, RGS2, p63ΔN, RGS-LSCII, or Gαt, as indicated. The cells were serum starved for 20 hours and stimulated with DMSO or 1 μM PGE2 for 24 hours. (C) NRVMs were infected with the 3xMEF2-Luciferase reporter and pretreated with the adenylyl cyclase inhibitor SQ22536 (100 μM) for 20 minutes or the Gi/o-protein inhibitor pertussis toxin (PTX, 100 ng/ml) for 20 hours in serum-starved conditions and treated with DMSO or 1 μM PGE2 for 24 hours.

Data information: In (B-D), n=3, independent experiments, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, RGS16, P<0.0001; RGS2, P=0.1725; p63ΔN, P=0.0666; RGS-LSCII, P=0.0818, SQ22536, P=0,385; PTX, P<0.0001; Gαt, P<0,0001), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 2.
Inflammation leads through PGE/EP3 signaling to HDAC5/MEF2-dependent transcription in cardiac myocytes (2018)
Figure 3

Figure 3 - PGE2 induces HDAC5 hyperphosphorylation and concomitant MEF2-activation via PKD.

A,B NRVMs were infected with recombinant adenovirus encoding Flag-HDAC5 (A) or not infected (B) and serum starved for 20 hours. The cells were pretreated with the PKC-inhibitor bisindolylmaleimide I (2 μM) or the PKD-inhibitor BPKDi (3 μM) for 20 minutes and stimulated with 100 nM endothelin-1 (ET1) or 100 μM phenylephrine (PE) or 1 μM PGE2 for 4 hours, as indicated. HDAC phosphorylation was detected by immunoblotting with the anti-HDAC5 phospho-Ser-498 antibody, phosphorylation of PKD was assessed using anti-phospho-PKD antibodies (Ser-744/Ser-748 and Ser-916), representative images are shown from 3 independent experiments.

C NRVMs were infected with the 3xMEF2-Luc reporter and serum-starved for 20 hours. The cells were pretreated with the PKD-inhibitor BPKDi (3 μM) or the CamKII-inhibitor AIP (1 μM) for 20 minutes and stimulated with DMSO or 1 μM PGE2 for 24 hours. N=3, * represents significant interaction between the two treatments (P<0.05, Two-Way ANOVA, BPKDi, P<0.0001; AIP, P=0.2197), values are mean±s.e.m. The exact n and P values can be also found in the Source Data excel file for Figure 3.
Inflammation leads through PGE/EP3 signaling to HDAC5/MEF2-dependent transcription in cardiac myocytes (2018)
Figure 4

Figure 4 - PGE2 induces Rac1 and MEF2 activation via Gi/o and the RacGEF Tiam1.

A NRVMs were serum-starved for 24 hours and stimulated for 2 minutes with 10 μM PGE2. Rac1 and RhoA GTPase activation was determined by effector pulldown assay and analyzed by immunoblot.

B NRVMs were infected with recombinant adenoviruses encoding EGFP (AdGFP) or RGS16 24 hours prior to stimulation, then Rac1 activity was measured,

C Cells were pretreated with 100 ng/ml pertussis toxin (PTX, Gi/o-inhibitor) for 24 hours or with 50 μM NSC23766 (NSC, Tiam1-Rac1 interaction inhibitor) for 2 hours. Thereafter Rac1 activation was determined by effector pulldown assay.

D NRVMs were transduced with the 3xMEF2-Luc reporter and were serum-starved for 20 hours. After 2-hour of 50 μM NSC23766 (A) pretreatments, the cells were stimulated with DMSO or 1 μM PGE2 for 24 hours, then MEF2 activity was determined. NSC abolished the MEF2 activation by PGE2.

Data information: In (A-C) representative images from at least 3 independent experiments are shown. In (B-D) values are mean±s.e.m., (B,C) n=4; (D), n=3; *,P<0.05 (Two-Way ANOVA, RGS16, P=0.0415 (B); PTX, P=0.039 (C), NSC effect on Rac1 activation, P=0.0412 (C); NSC effect on MEF2 induction, P<0.0001. PGE2 effect was significant in Rac1 activation, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 4.
Inflammation leads through PGE/EP3 signaling to HDAC5/MEF2-dependent transcription in cardiac myocytes (2018)
Figure 5

Figure 5 - PGE2 activates MEF2 through PAK2 in NRVMs.

A After serum starvation NRVMs were pretreated with or without 50 μM NSC23766 for 2 hours and stimulated with 1 μM PGE2 for 1 hour. Total and phosphorylated PKD levels were analyzed by immunoblot. PKD was activated by PGE2, but it was not affected by NSC.

B After infection with Flag-HDAC5 and serum starvation, NRVMs were treated with 50 μM NSC23766 for 2 hours prior to stimulation. The cells were stimulated with 1 μM PGE2 for 1 hour. HDAC5 phosphorylation at Ser-498 was detected by immunoblot. The phosphorylation of HDAC5 at this site was not prevented by NSC.

C NRVMs and neonatal cardiac fibroblasts (CFs) were serum-starved, pretreated with vehicle or 200 nM L798106 (EP3 receptor antagonist) for 20 minutes, and were stimulated with 1 μM PGE2 for 10 minutes. PAK2 and PKD phosphorylation was assessed by immunoblot. Calsequestrin was used as a myocyte-marker.

D MEF2 activity was determined in cells infected with the 3xMEF2-Luc reporter after serum starvation. Cells were stimulated with vehicle or 1 μM PGE2 for 24 hours after 1-hour of 30 μM IPA-3 pretreatment. IPA-3 inhibited the MEF2 activation by PGE2.

Data information: In (A-C) representative images from 3 independent experiments are shown. In (D) values are mean±s.e.m., n=3, biological replicates, *,P<0.05 (two-way ANOVA, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 5.
Inflammation leads through PGE/EP3 signaling to HDAC5/MEF2-dependent transcription in cardiac myocytes (2018)
Figure 6

Figure 6 - PGE2-induced nuclear export of HDAC5 is regulated by both PKD and PAK2

Top: GFP-HDAC5 infected NRVMs were serum-starved for a day, pretreated for two hours with the indicated compounds (BPKDi, 3 µM, IPA-3, 30 µM) alone or in combination, then were stimulated for 4 hours. Representative images. Scale bar is 10 µm. Bottom : Statistical analysis of cellular localization of HDAC5. GFP-HDAC5 localization (nuclear or cytosolic) was assessed in >100 cells of each sample (n=3, technical replicates). *, P<0,05 (one-way ANOVA with Bonferroni post-hoc test, vehicle vs. ET1 or vs. PGE2, P<0.0001; PGE2 vs. PGE2+BPKDi, P=0.0004; PGE2 vs. PGE2+BPKDi, P=0.0114; PGE2 vs. PGE2+BPKDi+IPA-3, P<0.0001). The exact n and P values can be also found in the Source Data excel file for Figure 6.
Inflammation leads through PGE/EP3 signaling to HDAC5/MEF2-dependent transcription in cardiac myocytes (2018)
Figure 7

Figure 7 - LPS-induced endotoxemia leads to myocardial inflammation, increased myocardial PGE2-levels and MEF2-activation.

MEF2-lacZ reporter mice (BALB/c-background, 6-12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 hours.

A mRNA-levels of different inflammatory cytokines, as indicated. The graphs show relative mRNA-levels, fold increase compared to saline-treated controls, normalized to 18s-content.

B PGE2 was quantified after mechanical homogenization of deeply frozen hearts using nano-liquid chromatography tandem mass spectrometry.

C Histologic and macroscopic stainings of β-galactosidase-activity in MEF2-lacZ reporter mice show MEF2-activation (blue cells and precipitates, respectively) in the myocardium upon LPS-treatment. Saline-treated littermates served as control. Scale bar of histological stainings is 100 µm. Quantification of whole-heart stainings is shown in the right panel (pixel intensity in blue channel normalized to total intensity).

D Induction of PKD, HDAC5 and PAK2 in myocardial inflammations. Immunoblots were performed on extracts of hearts of saline- and LPS-treated mice. Representative blots are shown in the left panel, the quantification is in the right panel.

Data information: values are mean±s.e.m. The exact n values are shown in the bottom of the bar graphs. *,P<0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s.=not significant. In (C) representative images of myocardial sections are shown from 3 independent experiments and representative whole hearts of 6 independent experiments. P values: IL6, P=0.0007 (A); TNFα, P<0.0001 (A); PGE2, P=0.0016 (B); blue pixel intensity, P=0.0083 (C); phospho-PKD-Ser-744/Ser-748, P=0.0049 (D); phospho-PKD-Ser-916, P=0.2684 (D); phospho-HDAC5-Ser-498 (D), P=0.0019; phospho-HDAC5-Ser-259, P=0.0003 (D); phospho-PAK2-Ser-192/Ser-197, P=0.1962 (D); phospho-PAK2-Thr-402, P=0.0446 (D). The exact n and P values can be also found in the Source Data excel file for Figure 7.