Anti lc3 1 2 antibody

The extraction of total protein from the treated cells was carried out using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Nantong, China). The concentration of protein was quantified with bicinchoninic acid (BCA) protein measurement kit (Thermo Fisher Scientific, USA). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA). The membrane was blocked with 5% skim milk at room temperature for 2 h, and then incubated with anti-GRP78 antibody, anti-PERK antibody, anti-Beclin1 antibody, anti-LC3 I/II antibody, and anti-GAPDH antibody (Cell Signaling Technology, USA) at 4 °C overnight. Next day, the relative secondary antibody was added, and the membrane was incubated at room temperature for 1 h. Then, enhanced chemiluminescence (ECL) reagent was added, and the membranes were detected by chemiluminescence (GE Healthcare, Piscataway, NJ, USA).

For immunofluorescence, patients’ and controls fibroblasts were seeded at the density of 20 x 10³ in 24-well cluster plates onto 12-μm cover glasses. After 24 h of culture in complete medium, cells were fixed with 3% PFA (30 min at 4°C) or absolute chilled methanol for 10 min at −20°C. After permeabilization with 0.5% TritonX-100 (10 min at room temperature), fibroblasts were stained with mouse monoclonal anti-Lamp1 antibody (Cell signaling), and/or rabbit polyclonal TOM20 antibody (Santa Cruz) and rabbit polyclonal GM130 antibody (Abcam), followed by the appropriate secondary antibody (Life Technologies) and DAPI.
To promote the autophagosomes formation fibroblasts were treated with EBSS medium (Earle’s Balanced Salt Solution) with or without bafilomycin for 4 hours, fixed with absolute chilled methanol for 10 min at −20°C. Cells were stained with a rabbit polyclonal anti-LC3I/II antibody (Cell Signaling) followed by the appropriate secondary antibodies (Life Technologies) and DAPI.