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. Author manuscript; available in PMC: 2021 Oct 1.
Published in final edited form as: FASEB J. 2020 Aug 24;34(10):13900–13917. doi: 10.1096/fj.202001515R

Figure 2: Endogenous PKI proteins modulate ERK phosphorylation.

Figure 2:

A. RT-qPCR analysis of endogenous PKI mRNAs relative to PKIA expression. Results shown are normalized means ± SD, N = 3. B. RT-qPCR of PKI mRNAs in cells transfected with siRNA for individual PKIA, PKIB, and PKIG, or in combination (siPKIABG). Expression relative to non-targeting control siRNA (siCon) transfected cells. Results shown are normalized means ± SD, N = 3, and analyzed with two-way ANOVA with Tukey’s post-hoc multiple comparisons test. C. Western blot panel of PKA-phosphorylated substrates after each siRNA transfection in cells with (+) or without (−) FSK and IBMX for 30 min. D. Western blot panel of indicated targets after depletion of endogenous PKIs and stimulated with (+) or without (−) FSK and IBMX for 30 min. E. CRE-Luc assay in cells treated with DMSO (Con) or FSK+IBMX for 6 hrs in control and PKI depleted cells. Results shown are normalized means ± SD, N = 5, and analyzed with two-way ANOVA with Tukey’s post-hoc multiple comparisons test. F. IF image showing localization of GFP-tagged PKIs. G. Western blot of PKA-phosphorylated substrates in cells transduced with or without PKIs and stimulated with (+) or without (−) FSK and IBMX for 30 min. H. Western blot panel showing changes to cAMP-mediated phosphorylation of target proteins in cells expressing full-length PKIs and stimulated (+) or not (−) with FSK and IBMX for 30 min. I. CRE-Luc assay showing alterations to PKA-mediated transcriptional activity in cells transduced with full-length PKIs or GFP as a control, then treated with DMSO (Con) or FSK+IBMX for 6 hrs. Results shown are normalized means ± SD, N = 6, and analyzed with two-way ANOVA with Tukey’s post-hoc multiple comparisons test. For all panels: **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not-significant p>0.05.