Anti sqstm1 p62 ab56416

After 24 h treatment with PD at various concentrations or 30 μM chloroquine (autophagy positive control), cells were washed in PBS and fixed with 4% paraformaldehyde (PFA) solution and permeabilized with 0.1% TRITON -X/PBS solution, then was performed a blocking in 1% BSA for 1 hat RT. Cells were incubated with LC3B antibody (Cell Signalling, USA) and Anti-SQSTM1/p62 (ab56416, Abcam, Cambridge, UK) in PBS for 30 min. Secondary antibodies were added after a PBS wash in the same conditions. Cells were incubated in a 1:500 solution of 10 mg/mL Hoechst (Invitrogen) in PBS for 10 min in the dark. To stain endoplasmic reticulum cells were incubated with 200 nM ER-Tracker Blue-White DPX in PBS solution for 20 min at 37 °C. For positive control cells were exposed for 16 h to 5 μg/mL tunicamycin. Images were collected under a fluorescence microscope (EVOS FL Cell Imaging System, Thermo Scientific, Rockford, USA). To stain lysosomes, cells were incubated with 60 nM LysoTracker (Thermo Fisher Scientific, USA) for 45 min at 37 °C. ImageJ (Fiji plugin) software was used for the calculation of Puncta and Intensity correlation quotient (ICQ).

Chloroquine (CQ) and 3‐methyladenine (3‐MA) were purchased from Sigma‐Aldrich Corp. (St. Louis, MO, USA). Dulbecco's Modified Eagle Medium: Nutrient Mixture F‐12 (DMEM‐F12) media, foetal bovine serum (FBS), mouse epidermal growth factor (EGF), Alexa Fluor 488‐ and Alexa Fluor 594‐conjugated IgG were purchased from Life Technologies (Carlsbad, CA, USA). Rapamycin (Rapa), the antibodies of anti‐LC3B (ab63817), anti‐Beclin1 (ab55878) and anti‐SQSTM 1/p62 (ab56416) were purchased from Abcam (Cambridge, CS, UK). The antibodies specific for PERK (3192), phospho‐PERK (Thr980, 3179), mTOR (2983), phospho‐mTOR (Sre2448, 2971), AKT (4691), phospho‐AKT (Sre473, 9271), phosphor‐ULK1 (Ser555, 5869) and CHOP (2895) were purchased from Cell Signaling Technology (Boston, MA, USA). Salubrinal (Sal) was purchased from Amquar Bio Corp. (Denver, CO, USA). Anti‐β‐actin antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Inserts used in this study were purchased from Merck Millipore (Bedford, MA, USA). Plastic cell culture dishes and six‐well plates were purchased from BD Biosciences (Lincoln Park, NJ, USA).