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. 2022 Jun 10;298(7):102121. doi: 10.1016/j.jbc.2022.102121

Figure 8.

Figure 8

RasGRP3 is involved in PKCα-induced growth inhibitory ERK signaling.A, i, Western blot analysis confirms that IEC-18 cells do not express RasGRP1. SW480 and FET colon cancer cells and Q21.43 patient-derived colon cancer organoids are used as positive controls for expression of RasGRP1. ii, IEC-18 cells were transfected with NT siRNA or ON-TARGETplus SMARTpool siRNA targeting RasGRP1 and/or RasGRP3 as indicated 24 h prior to treatment with PMA for 15 min. The graph under the blots shows densitometric analysis of relative levels of pERK normalized to loading control (±s.d., n = 3. ∗p ≤ 0.05). iii, as in (ii), except that cells were treated with PMA for 2 h. B, IEC-18 cells were transfected with NT or ON-TARGETplus SMARTpool RasGRP3-targeted siRNA and treated for 15 min with PMA (in full serum), or with EGF (after serum-starvation), before Western blot analysis for the indicated proteins. The data are from single immunoblots; vertical lines are included for clarity. C, i, IEC-18 cells were treated with 100 nM PMA for 15 min before lysis and treatment with lambda phosphatase (λPP) as indicated. Lysates were run on Phos-tag gels and analyzed by immunoblotting. ∗ Slower mobility RasGRP3 species induced by PMA treatment. Data are from a single membrane; the vertical line indicates rearrangement of the blot for clarity. ii, IEC-18 cells were pretreated with 4 μM Gö6976 to inhibit PKCα activity, followed by 15 min treatment with 100 nM PMA as indicated. Cells were then lysed and lysates were divided and treated with lambda phosphatase as indicated prior to Phos-tag gel/immunoblot analysis. ∗ As in C(i). D, nontargeting (NT) and RasGRP3 siRNA transfected cells were treated with vehicle or PMA as in (A) before lysis and isolation of active GTP-bound Ras by GST-Raf1-RBD pulldown. Levels of the indicated proteins in the original lysates and Raf1-RBD pulldowns (Total Ras pulldown) were detected by Western blotting. Detection of Total Ras used a pan-Ras antibody that targets all three Ras proteins. The panel to the right shows the signal obtained following pulldown of GDP- and GTP-loaded Ras as negative and positive controls, respectively. All data are representative of at least three independent experiments. PMA, phorbol 12-myristate 13-acetate.