Figure 2
Western blot analysis for ERK and MEK activation. The indicated CRC cell lines were cultured, cells harvested, extracts prepared and western blots performed. (a) Cells (HCT116, SW620 and KM12L4A) cultured with ISP, an inhibitor of PTPRS, or a scrambled control peptide (SC). Western blots to detect ERK, tyrosine phosphorylated ERK, MEK and phosphorylated MEK, and AKT and phosphorylated AKT are shown as indicated. The quantitations were determined by normalizing the phosphorylated protein values with the total protein; then dividing the ISP values by the SC values. (b) The indicated cells had PTPRS knocked down with siRNA to PTPRS (siPTPRS) or were treated with a scrambled siRNA control (siCtl). Western blot analysis shows PTPRS, phospho-ERK, ERK, phospho-MEK, MEK, phospho-AKT, AKT, and alpha-tubulin. Knockdown of PTPRS via siRNA shows results consistent with the ISP treatments. (c) CRISPR knockouts of PTPRS in HCT116, SW620, and KM12L4A cell lines and their CRISPR control cell lines where cell extracts were used in western blot analysis for phosphorylation of ERK and MEK. This analysis shows PTPRS, ERK, phospho-ERK, MEK, phospho-MEK, AKT, phospho-AKT, and alpha tubulin in the cell line pairs (Ctl and KO) as indicated. (d) ddPCR analysis of PTPRS expression in CRISPR KO cells for HCT116, SW620, and KM12L4A. Analysis of the ddPCR result shows a near complete knockout for HCT116 and KM12L4A; SW620 shows >85% knockout. All experiments were done in triplicate. The mean and standard deviation are shown. Two-tailed, paired t test was used to determine the statistical significance for comparison as indicated.
