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Cells were subsequently blocked by 2% BSA in PBS-T (0.1% Tween 20 in PBS) for 1 h at room temperature, incubated with the primary antibody Anti-Rheb (Santa Cruz Biotechnology Cat# sc-271509, RRID: AB_10659102, 1:500) overnight at 4 °C in blocking buffer, then washed with PBS-T three times and incubated with Alexa-555-conjugated secondary antibody (Invitrogen # A-31570, RRID:AB_2536180, 1: 1000) and DAPI (1 µg/ml) in blocking buffer for 1 h at room temperature with protection from light