Figure 2(A) Western blot analysis of Protein Kinase A phospho-substrates (PKA Ⓟ-subs) in PCBP1 immunoprecipitates from HeLa cells exposed to sequential stimulation (same treatment in both treatment days) with forskolin (Fsk) in the absence or presence of the PKA inhibitor PKI14-22 (PKI). Reprobing with anti-PCBP1 antibodies is shown in the middle blot. Lower blot shows the total amount of PCBP1 in the cell lysates. Quantification of three independent experiments is shown on the right. Results are expressed as average ± SEM of fold increase over vehicle normalized to PCBP1 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(B) Nuclear localization of PCBP1 analyzed by Western blotting of extracts from HeLa cells exposed to sequential stimulation (same treatment in both treatment days) with activin A (Act), forskolin (Fsk), PKA inhibitor (PKI) and the ALK4 inhibitor SB431542 (SB) as indicated. Blots of nuclear and cytosolic fractions were reprobed with anti-PCNA and anti-αTubulin (αTub) antibodies, respectively. Quantification of three independent experiments is shown on the right. Results are expressed as average ± SEM of fold increase over vehicle (first column) normalized to PCBP1 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(C) Western blot analysis of Smad3 in PCBP1 immunoprecipitates from HeLa cells exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), forskolin (Fsk) or their combination as indicated. The Act+Fsk extracts were also immuoprecipitated using control IgG antibodies as additional control. Reprobing with anti-PCBP1 antibodies is shown in the middle blot. The lower blot shows unaltered Smad3 levels across all conditions. Quantification of four independent experiments (biological replicates) is shown on the right. Results are expressed as average ± SEM of fold increase of Smad3/PCBP1 co-immunoprecipitation (coIP) over vehicle (first column) normalized to PCBP1 levels. (D) Western blot analysis of PCBP1 in Smad3 immunoprecipitates from HeLa cells using conditions identical to panel (C). The lower blot shows unaltered PCBP1 levels across all conditions. Quantification of four independent experiments (biological replicates) is shown on the right. Results are expressed as average ± SEM of fold increase of PCBP1/Smad3 co-immunoprecipitation (coIP) over vehicle (first column) normalized to Smad3 levels. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(E) Representative immunofluorescence staining of Smad3 (green) and PCBP1 (red) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM.(F, G) Quantification of PCBP1+ (F) or Smad3+ (G) nuclei in cultured MSNs after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying PCBP1 (F) or Smad3 (G) in the nucleus. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(H) Quantification of PCBP1/Smad3 co-localization in nuclei of cultured MSNs using conditions identical to panel (F). Results are expressed as average ± SEM (biological replicates) of % of MAP2+ cells displaying co-localization of PCBP1 and Smad3 in the nucleus. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(I) RT-qPCR analysis of PCBP1 mRNA in cultured MSNs exposed to consecutive stimulation as indicated (same treatment in both treatment days). Results are expressed as average ± SEM of fold change over vehicle. No significant differences were found by one-way ANOVA followed with Tukey's multiple comparisons test (p≥0.05; N=4). Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(J) Representative images of PLA (red) between Smad3 and PCBP1 (S3/PCBP1) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF), PKA inhibitor (PKI) or SB431542 (SB) as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM. (K) Quantification of PLA between Smad3 and PCBP1 in cultured MSNs after the indicated treatments. Results are expressed as average ± SEM of number of PLA dots per cell. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.