Fig. 5. Slack is basally regulated by p38 MAPK via phosphorylation at S736 and S742.

(a) Representative western blot of Slack protein from Slack stable HEK cell lysates. Pull-down using a phospho-specific Serine-Proline antibody was followed by western analysis using a Slack-specific monoclonal antibody. (b) Representative blot of Slack protein in Slack Stable HEK cells, as detected by western blotting using Slack antibody following pull-down by the pS-P antibody. Cells were untreated or treated with either Vehicle (0.01% DMSO) or p38 Inhibitor (10 μM) for 90 minutes at 37°C. (c) Representative blot of Slack protein in CHO cells transiently transfected with either wild type (WT) (‘-‘ and ‘+’ denoting IPs done in the absence and presence of pS-P antibody, respectively) or mutant Slack pTRACER expression plasmid in which Serines 736 and 742 were both replaced with Alanine. Cells were subjected to western analysis for Slack following pull-down using the pS-P antibody. (d) Top- Representative traces of whole-cell recordings from CHO cells transiently transfected with either WT (left), double alanine mutated (middle) or double glutamate mutated (right) Slack pTRACER. Bottom- Current voltage relationship of I_K measured from WT, S736A+ S742A or S736E + S742E Slack pTRACER- transfected CHO cells. Holding potential was −70 mV, and and currents were elicited with voltage steps from −120 mV to +120 mV in 20 mV increments. Data is expressed as mean +/− SEM (n=10 for all groups). Statistics performed using One Way ANOVA followed by multiple comparisons using Tukey’s method (*p<0.05 between WT Slack and S736A + S742A groups, ^#p<0.05 between WT Slack and S736E + S742E groups).