CS1 knockdown affects phosphorylation of ERK1/2, STAT3, and AKT. (A) CS1 knockdown after lentiviral CS1 shRNA infection. RNA was prepared from OPM2 cells infected with individual lentiviral CS1shRNA (targeting 4 regions on CS1 cDNA) or control shRNA, followed by gene expression analysis. Arbitrary unit of CS1 mRNA is shown. Immunoblotting and flow cytometric analysis further confirmed CS1 membrane depletion in CS1null OPM2 cells. Solid histogram represents CS1 expression; open histogram, isotype control. (B) Phosphorylated ERK1/2, STAT3, and AKT in C1null OPM2 versus cntOPM2 cells were examined using specific phosphorylated Abs. α-Tubulin was used as a loading control. (C) Cell lysates were examined for the phosphorylation status of more than 37 phospho-sites in 29 signaling proteins with a panel of 37 highly validated phospho-site-specific Abs (Kinetworks KPSS-1.3 Phosphosite Screen, Kinexus Biosystems). Representative multiple immunoblots and the migration of target phosphoproteins are shown. (D) The intensity of the ECL signals (counts per minute) was quantified from the multiple immunoblots for the total cell lysates of CS1null OPM2 and cntOPM2. The fold changes of phosphorylation in the CS1null OPM2 (■) relative to cntOPM2 (□) are presented. The averaged results from 2 phospho-site analyses are shown.