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. 2020 Jan 28;11:562. doi: 10.1038/s41467-019-14259-y

Fig. 4. HSP90A contributes to NANOG-induced AKT activation through TCL1A-stabilization.

Fig. 4

a–e CaSki-NANOG cells were transfected with siGFP or siHSP90AA1-#1. a Levels of HSP90A, TCL1A, pAKT, AKT, MCL1, and Cyclin A were analyzed by immunoblotting. Graph depicts the experimental quantitation based on at least three independent experiments. **p < 0.001, ***p < 0.0001 by two-tailed Student’s t test. NS not significant. b The cells were treated with cycloheximide (CHX) for the indicated times. Cell lysates were subjected to immunoblotting with anti-TCL1A antibodies. Graph represents the means ± SD of three quantified data, after normalization to the corresponding β‐ACTIN level. c The cells were treated with or without MG132 (10 μM) for 8 h. Cell lysates were subjected to immunoblotting with anti-HSP90A and anti-TCL1A antibodies. d The cells, transfected with indicated plasmids, were treated with MG132. Cell lysates were immunoprecipitated with anti-Myc antibody, and immunoblotted with the anti-HA antibody. The input represents 5% of total proteins. e siHSP90AA1-transfected CaSki-NANOG cells were transfected with TCL1A-Myc constructs. Levels of HSP90A, TCL1A, pAKT, and AKT were analyzed by immunoblotting. f HEK293 cells were transfected with TCL1A-Myc. g TCL1A-Myc-transfected HEK293 cells were treated with DMSO or AUY-922 as indicated. f and g Cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blotting using anti-Myc and anti-HSP90A antibodies. h HEK293 cells, transfected with indicated plasmids, were treated with or without MG132 (10 μM) for 8 h and were subjected to immunoblotting with anti-Myc and anti-HSP90A antibodies. Graph depicts the experimental quantitation of TCL1A protein level based on at least three independent experiments. The p-value by one-way ANOVA is indicated. i CaSki-NANOG cells were transfected with HA-Ub or TCL1A-Myc constructs and then were treated with DMSO or AUY-922 as indicated. Cell lysates were immunoprecipitated with anti-Myc antibody, and immunoblotted with the anti-HA antibody. The input represents 5% of total proteins. Numbers below blot images indicate the expression as measured by fold change a–c, e and h. All experiments were performed in triplicate. Data represent the mean ± SD a and h. Source data are provided as a Source Data file.