Figure 5. GABARAPL1 knockdown leads to decreased LAMP1 protein levels and immunocytochemistry staining.(A) MDA-MB436-shC, sh2, and sh5 cells were cultured for 24 h at 37 °C and 5% CO₂ then total proteins (25 µg) were separated on 12% SDS-PAGE gels followed by immunoblotting with anti-LAMP1 antibodies and the ECL Plus reagent. A representative experiment of 3 performed is shown. (B) Quantification of the signals observed on the western blot in (A). *P < 0.05, vs shC (n = 3). (C) GABARAPL1 and LAMP1 mRNA expression was analyzed by qRT-PCR in the MDA-MB436-shC and sh2 cells. *P < 0.05, vs shC. (D) MDA-MB436-shC, sh2 and sh5 cells were cultured for 24 h at 37 °C and 5% CO₂, fixed, permeabilized, blocked with 5% BSA, incubated with a monoclonal anti-mouse LAMP1 overnight at 4 °C and then with an Alexa Fluor 555 goat anti-mouse for 1 h. The cells were then analyzed using a confocal microscope. Each picture is representative of a typical cell staining observed in 10 fields chosen at random.