Lc3b rabbit polyclonal antibody

Frozen lung tissues were sonicated in RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 1 mM EDTA) containing protease inhibitor cocktail (Roche Applied Bioscience). Protein content was determined by Bradford assay (Amresco) staining using bovine serum albumin as a standard. Equal amounts of protein were loaded on 10%, 12%, or 15% Tris-glycine gels for electrophoresis. Proteins were wet-transferred to PVDF membranes (Millipore) and then probed with the indicated antibodies. The primary antibodies and dilutions used were as follows: anti-surfactant protein B rabbit polyclonal antibody (1:3,000, #07-614, Millipore), anti-surfactant protein C rabbit polyclonal antibody (1:1000, #AB3786, Millipore), anti-surfactant protein A rabbit polyclonal antibody (1:1000, #AB3420, Millipore), anti-p62 rabbit polyclonal antibody (1:1000, #P0067, Sigma), LC3B rabbit polyclonal antibody (1:1000, #12741, Cell Signaling Technology), anti-ATG7 rabbit polyclonal antibody (1:1000, #A2856, Sigma) and anti-actin mouse polyclonal antibody (1:1000, #TA-09, Zsgb-bio, China). Immunoreactivity was detected using horseradish peroxidase-conjugated secondary antibodies. Chemiluminescence substrates were used (Tiangen, Beijing, China). The images were captured using a MicroChemi 4.2 system (DNR Bio Imaging Systems, Jerusalem, Israel).

AGS-BDneo and HA cells grew on cover slips were treated with drugs for specific duration depending on experimental needs. Cells were fixed with acetone for 10 mins at room temperature. The fixed cells were then stained with anti-Zta, cleaved caspase-3, or LC3B rabbit polyclonal antibody (1:200; Cell Signaling Technology, Beverly, MA) overnight at 4 °C. Expression of the proteins was visualized with Alexa Fluor 488 F(ab′)2 fragment of goat anti-rabbit IgG antibody (1:500; Invitrogen) under fluorescence microscopy or Carl Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). Nuclei of cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Roche, Mannheim, Germany).

HONE-1 and HA cells grew on cover slips were treated with drugs for specific duration depending on experimental needs. Cells were first washed with PBS once and were fixed with ice-cold acetone for 10 min at room temperature. The fixed cells were then washed with PBS twice and were blocked with 5% normal goat serum (PCN5000, Gibco, Gaithersburg, MD, USA) in 1× TBST (50 mM Tris, 150 mM NaCl, 0.1% Triton-X, pH 7.4) for 30 min at room temperature. After that, the cells were stained with anti-Zta monoclonal antibody (1:50), LAMP-1, cleaved caspase-3, or LC3B rabbit polyclonal antibody (1:200; Cell Signaling Technology, Beverly, MA, USA) in 5% normal goat serum, 1× TBST overnight at 4 °C. Expression of the proteins was visualized with Alexa Fluor 594 F(ab′)2 fragment of goat anti-mouse IgG antibody or Alexa Fluor 488 F(ab′)2 fragment of goat anti-rabbit IgG antibody (1:500; Invitrogen) under fluorescence microscopy. Nuclei of cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Roche, Mannheim, Germany).