Figure 4. GABARAPL1 knockdown leads to increased SQSTM1 and BECN1 proteins.(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-BECN1, anti-SQSTM1 and anti-ACTIN 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, GABARAP, LC3B, BECN1, and SQSTM1 mRNA expression was analyzed by qRT-PCR in the MDA-MB436-shC, and sh2 cells. *P < 0.05, vs shC (n = 3). (D) MDA-MB436-shC and sh2 cells were cotransfected with the vectors expressing HA-SQSTM1 and pEGFP-N1 (ratio 10:1). Forty-eight hours after transfection, total proteins (25 µg) were separated on 12% SDS-PAGE gels, followed by immunoblotting with anti-HA, anti-GFP, and anti-ACTIN antibodies and the ECL Plus reagent. A representative experiment of 3 performed is shown. (E) Quantification of the signals observed on the western blot in (D). *P < 0.05, vs shC (n = 3).