Fig. 4 | Signal Transduction and Targeted Therapy

Fig. 4

From: B-lymphoid tyrosine kinase-mediated FAM83A phosphorylation elevates pancreatic tumorigenesis through interacting with β-catenin

Fig. 4

FAM83A is phosphorylated by BLK kinase at Y138. a The mass spectrum showed that FAM83A extracted from HEK293T cell lysates was phosphorylated at tyrosine 138 residue. b In vitro purified GST-FAM83A were incubated with HEK293T cell lysates with or without ATP addition, and the reaction products were subjected to western blotting. c In vitro purified GST-tagged FAM83A and GST-tagged FAM83A Y138A mutant were incubated with HEK293T cell lysates with or without ATP addition, and the reaction products were subjected to western blotting (n = 3). d The in vivo interaction between Flag-tagged FAM83A and the components of non-receptor TEC kinase subfamily or SRC kinase subfamily were analyzed in HEK293T cells by western blotting (n = 3). e Level of total tyrosine phosphorylation of Flag-tagged FAM83A and its Y138A mutant in HEK293T cells after HA-tagged BTK, FYN, BLK, and HCK transfection. Cell lysates were used for IP and western blotting with the indicated antibodies (n = 3). f Level of total tyrosine phosphorylation of Flag-tagged FAM83A and its Y138A mutant in HEK293T cells after BLK kinase knockdown using two specific small interfering RNAs (siBLK#1 and siBLK#2). Cell lysates were used for IP and western blotting with the indicated antibodies (n = 3). g, h Level of total tyrosine phosphorylation of Flag-tagged FAM83A and its Y138A mutant in HEK293T cells with or without BLK kinase overexpression and saracatinib (BLK inhibitor, 10 μM)/PP2 (LCK or FYN inhibitor, 10 μM) treatment. Cell lysates were used for IP and western blotting with the indicated antibodies (n = 3). i The BLK kinases was obtained from the immunoprecipitate of HEK293T cell lysates that transfected with either HA-tagged BLK wild-type, BLK kinase-dead mutant (Y389A) or BLK constitutive activated mutant with the deletion of the C-terminal auto-inhibitory domain (BLK ΔC) using HA tag antibody. Then the BLK kinases were incubated with the in vitro purified GST-tagged FAM83A protein at 30 °C for 30 min with or without BLK inhibitor saracatinib, and then the reaction products were subjected to western blotting assay (n = 3)

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