Fig 1: The de-ubiquitination of Vimentin by USP14 depends on the expression of BECN1. (a) H1299 cells were transfected with pCMV-HA-USP14 plasmid. Forty-eight hours later, HA-USP14 was immunoprecipitated using rabbit anti-HA antibody followed by Western blot. BECN1 was detected using mouse anti-BECN1 antibody and HA-USP14 was detected using mouse anti-HA antibody. (b) H1299 cells were transfected with pCMV-HA-BECN1 plasmid. Forty-eight hours later, HA-BECN1 was immunoprecipitated using mouse anti-HA antibody followed by Western blot. USP14 was detected using rabbit anti-USP14 antibody and HA-BECN1 was detected using rabbit anti-HA antibody. (c) H1299 cells were transfected with pCMV-HA-BECN1 plasmid. Forty-eight hours later, immunoprecipitation was performed using normal rabbit IgG and rabbit anti-USP14 antibody followed by Western blot. USP14 was detected using mouse anti-USP14 antibody and Vimentin was detected using mouse anti-Vimentin antibody. (d) H1299 cells were transfected with BECN1-specific siRNA. Forty-eight hours later, immunoprecipitation was performed using normal rabbit IgG and rabbit anti-USP14 antibody followed by Western blot. USP14 was detected using mouse anti-USP14 antibody and Vimentin was detected using mouse anti-Vimentin antibody. (e) H1299 cells were transfected with pCMV-HA-USP14 alone or co-transfected with pCMV-HA-USP14 and pCMV-HA-BECN1. Forty-eight hours later, immunoprecipitation was performed using mouse anti-Vimentin antibody. The K48-linked ubiquitination was detected using K48-linkage specific polyubiquitin antibody. Vimentin was detected using rabbit anti-Vimentin antibody. (f) H1299 cells were transfected with pCMV-HA-USP14 alone or co-transfected with pCMV-HA-USP14 and BECN1 siRNA. Forty-eight hours later, immunoprecipitation was performed using mouse anti-Vimentin antibody. The K48-linked ubiquitination was detected using K48-linkage specific polyubiquitin antibody. Vimentin was detected using rabbit anti-Vimentin antibody.