Supplementary Methods and Figures 1 - 11 from BMX Negatively Regulates BAK Function, Thereby Increasing Apoptotic Resistance to Chemotherapeutic Drugs

<p>Supplementary Figure S1. Controls for co-immunoprecipitation of BAK-BMX complex. Supplementary Figure S2. Immunoprecipitation of BMX protein from HT1080 cells did not detect BAX associated with BMX. Input for immunoprecipitation reaction is 10% starting protein. Supplementary Figure S3. Representative images of proximity ligation in situ assay (PLISA) in HT1080 cells. Supplementary Figure S4. Immunoprecipitation of BMX protein from HT1080 cells used in in vitro kinase assay (Figure 1E). Input for immunoprecipitation reaction is 10% starting protein. Supplementary Figure S5. Representative images of BMX immunostaining in sections of prostate cancer tissue microarrays showing examples of low and high BMX reactivity in tumor tissue. Supplementary Figure S6. Immunoprecipitation of PTPN21 from HT1080 cells, then western blotted for BMX showing PTPN21-BMX interact in our model system. Supplementary Figure S7. Determination of PTPN21 knockdown in HT1080 cells following A) siRNA or B) shRNA treatment by qRT-PCR. Change in C(T) relative to the parental cells is plotted (for siRNA n=2, + range; for shRNA n=3, + SD). Supplementary Figure S8. A) Knockdown of PTPN21 increases BAK activation in response to UV damage. B) Knockdown of PTPN21 using 2 different shRNA sequences increases BAK activation in response to camptothecin damage. Supplementary Figure S9. Mean percentage of cells undergoing apoptosis as determined by Annexin V positivity by FACS analysis of HT1080 cells stably transfected with shRNA HuSH43 to knockdown PTPN21 expression over a 24 hour time course of CPT treatment (n=3, + SEM, * p<0.05). Supplementary Figure S10. Silencing of BMX expression in HT1080 cells. Supplementary Figure S11. BMX over-expression in HT1080 cells.</p>