Following treatments, cells were processed as described previously . Basically, cells were washed with PBS 1X and lysed in sample buffer (SB) 1X (2% (v/v) sodium dodecyl sulfate (SDS), 10% (v/v) glycerol, and 50 mM Tris–HCl, pH 6.8, in distilled water) by pipetting until homogenization. Protein concentration was measured based on the bicinchoninic acid assay, using bovine serum albumin as a standard. Samples were heated at 95 °C for 10 min before their quantification.
Equal amounts of protein (25–40 µg/condition) were resolved by 12% SDS-gel electrophoresis and transferred to polyvinylidene fluoride membranes, according to a partially modified conventional protocol . Immunodetection included transferring (15 V during 15 min, per each membrane) and blocking of the membrane with WB blocking solution (10% w/v fat free milk in Tris-buffered saline with Tween 20 (TBST)) for 1 h at room temperature. After washing the membranes 2 times with TBST 1X, blots were incubated with the corresponding primary antibody: p62/SQSTM1 (1:5000) (BD Transduction Laboratories, 610498), LC3-B (1:5000) (Sigma–Aldrich, L7543) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5000) (Millipore, MAB374) at 4 °C overnight, GAPDH (1 h at room temperature). The membranes were washed 2 times with TBST 1X and subsequently incubated with their respective HRP-conjugated secondary antibodies (1:10000) (Bio-Rad, 170–6515 and 170-5047 for rabbit and mouse antibodies, respectively) for 1 h at room temperature. Detection of bound antibodies was visualized by chemiluminescence using ECL substrate (Thermo Scientific, 32106). Quantification data analysis was performed using ImageJ software (NIH), establishing GAPDH protein levels as a loading control.
For the detection of endogenous p62 and LC3B, cells were seeded on coverslips, fixed with paraformaldehyde (4% in PBS 1X) and permeabilized with Triton X-100 solution (0.1% in PBS 1X) for 10 min. To block non-specific binding, cells were incubated with 10% FBS in PBS 1X for 20 min, followed by incubation with primary antibodies anti-p62 (1:500) and anti-LC3B (1:500) for 1 h at room temperature. After that, cells were incubated with Alexa Fluor 488 anti-rabbit (1:1000) (Molecular Probes, A-11034) and 568 anti-mouse (1:1000) (Molecular Probes, A-11004) secondary antibodies for LC3 and p62, respectively. Finally, coverslips were mounted on microscope slides, by using fluoromount-G (SouthernBiotech, 0100–01) medium. Images were taken by using an inverted fluorescence microscope (Olympus, IX-51) equipped with a camera (Olympus, DP70). The quantitative measurement of the fluorescence signal was performed using ImageJ software analyzing at least 200 cells per condition. Immunofluorescence procedure was developed as previously described . Full paper
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