Flotillin-1 promotes the progression of hepatocellular carcinoma by activating TFE3-mediated Golgi stress response via inhibition of mTORC1/2

Figure 5 Co-immunoprecipitation verified that flotillin-1 enhanced transcription factor E3 dephosphorylation and nuclear translocation by recruiting and inhibiting mechanistic target of rapamycin complex 1/2 protein and promoting its ubiquitination. A: Expression of flotillin-1 (FLOT1), mechanistic target of rapamycin (mTOR), and transcription factor E3 (TFE3) protein after immunoprecipitation (IP) (FALG-FLOT1) was detected by western blotting; 293T cells were divided into the Input, immunoglobulin G (IgG), and FLAG-FLOT1 groups. The Input group was the total protein sample without IP treatment after protein extraction; IgG group was the IgG isotype of FLOT1 antibody or TFE3 antibody as the negative control group; the FALG-FLOT1 group was the samples treated by IP with FLOT1 antibody. Immunoblotting (IB) was conducted using antibodies for detecting FLOT1, mTOR, and TFE3 proteins; B: Expression of FLOT1, mTOR, and TFE3 protein following IP with HA-TFE3. Again, 293T cells were divided into the Input (3 × HA TFE3), Input (3 × HA TFE3 + 3 × FLAG FLOT1), IgG (3 × HA TFE3 + 3 × FLAG FLOT1), IgG (3 × HA TFE3 + 3 × FLAG FLOT1), HA-TFE3 (3 × HA TFE3 + 3 × FLAG FLOT1), and HA-TFE3 (3 × HA TFE3 + 3 × FLAG FLOT1) groups. The Input group was the total protein sample without IP treatment after protein extraction; HA-TFE3 group comprised the samples treated by IP with TFE3 antibody; IgG group was the IgG isotype of TFE3 antibody as the negative control group; IB was conducted using antibodies for detecting FLOT1, mTOR, and TFE3 proteins; C: Expression of ubiquitin (Ub) protein after IP (mTOR) was detected by western blotting. Here also, 293T cells were divided into the control and 3 × FLAG-FLOT1 groups. IP was performed using the mTOR group as the sample antibody, while IB was conducted using Ub as the antibody for subsequent western blotting detection. FLOT1: Flotillin-1; TFE3: Transcription factor E3; mTOR: Mechanistic target of rapamycin.