Figure 5. Reconstitution of PMA/PKC mediated phosphorylation of Ser¹⁶⁷⁴ and Ser¹⁹²⁸ in HEK cells.

A, Recombinant WT α_1c was transiently co-expressed with β_2a and PKCβ1 in HEK293 cells. Cells were exposed to PMA (1 μM) and calyculin A (Cal: 50 nM) for 10 min. Bisindolylmaleimide (Bis, 0.5 μM) was pre-incubated for 1 hr at 37°C. Cells were harvested 24-48 hours after transfection, and lysed in the presence of phosphatase inhibitors. Lysates were size-fractionated on SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and blotted with anti-phospho-Ser⁵²⁸ (pS1928; upper panel) or α_1c (middle panel) antibodies. Arrowhead indicates size of anticipated phosphorylated band. Representative of 3 similar experiments. Lower panel, Activation of PKC by PMA had no effect on Ser⁵²⁸ phosphorylation. Mean ± SD. B, Recombinant WT or Ala-substituted Ser¹⁶⁷⁴ (S1674A) α_1c was transiently co-expressed with β_2a in HEK293 cells, in the absence or presence of PKCβ1 as indicated. Methodology is identical to (A) except nitrocellulose membranes were blotted with anti-phospho-Ser¹⁶⁷⁴ (pS1674; upper panel) or α_1c (middle panel) antibodies. The specificity of the pS1674 antibody is shown using the S1674A α_1c mutant. Representative of 3 similar experiments. Lower panel, Activation of PKC by PMA increased phosphorylation of α1c Ser¹⁶⁷⁴. Phosphorylation of Ser¹⁶⁷⁴ was caused by PKC because bisindolylmaleimide (Bis) prevented Ser¹⁶⁷⁴ phosphorylation. C, Recombinant WT or Ala-substituted Ser¹⁹²⁸ (S1928A) α_1c was transiently co-expressed with β_2a in HEK293 cells, in the absence or presence of PKCβ1 as indicated. Methodology is identical to (A) except nitrocellulose membranes were blotted with anti-phospho-Ser¹⁹²⁸ (pS1674; upper panel) or α_1c (middle panel) antibodies. Representative of 3 similar experiments. Lower panel, Activation of PKC by PMA increased phosphorylation of α1c Ser¹⁹²⁸. *, p<0.05