NPM-ALK interacts with PTPN1 and PTPN2. (A) Co-IP assay was performed on TS cells treated with 300 nM crizotinib for 3 hours. Co-IP was performed with anti-ALK antibody (IP ALK) or the corresponding isotype control (IgG) and analyzed by western blot probed with the indicated antibodies. One representative experiment out of 2 is shown. (B) Co-IP was performed on 293T cells transfected with NPM-ALK or the mock vector (-) and collected after 48 hours. Co-IP was performed with anti-ALK antibody (IP ALK) or the corresponding isotype control (IgG) and analyzed by western blot probed with the indicated antibodies. One representative experiment out of 2 is shown. (C-D) Representative images of in situ PLA (red) between NPM-ALK and PTPN1 (C) and NPM-ALK and PTPN2 (D) in TS and JB6 cell lines. WT TS and JB6 cells treated for 12 hours with TL13-112 100 nM were used as control to specifically reduced the anti-ALK antibody binding; PTPN1 KO and PTPN2 KO JB6 and TS cells were used as negative controls to remove anti-PTPN1 and PTPN2 antibody binding. (E) Quantification of PLA between ALK and PTPN1 and ALK and PTPN2 (C). Data are represented as mean ± SD; *P < .05, Student t test. TCL, total cell lysates.