Anti lc3 l7543

Cells were collected and lysed using RIPA buffer (150 mM NaCl, 1% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris-HCl pH 8) supplemented with protease and phosphatase inhibitors (Roche, Switzerland) and total protein concentration were measured by BCA assay. Whole-cell lysates (~20–30 μg of proteins) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblotting was performed using the following primary antibodies: anti-p38 (#8690), anti-phospho p38 (T180/Y182 - #4511), anti-p53 (#2524), anti-phospho pS6 (S235/236 - #4058), anti-Akt (#9272), anti-phospho-Akt (S473, #4060), anti-caspase-3 (clone 8G10; #9665), anti-HK2 (#2867), anti-HER2 (#2165), anti-mTOR (#2983), anti-phospho-mTOR (S2448—#5536), anti-PARP (#9542) from Cell Signaling Technology (Massachusetts, USA); anti-LC3 (L7543) from Sigma-Aldrich; anti-Actin (sc-8432), anti-GAPDH (sc-166545), anti-IκB-α (sc-371), anti-Lamp2 (sc-18822) and anti-tubulin (sc-73242) from Santa Cruz Biotechnology (Texas, USA); and anti-Hsc70 (10654–1-AP) from Proteintech (Illinois, USA).

The following primary antibodies were used in this study: anti-LRRK2 (MJFF2 [c41-2]; Abcam), anti-human LAMP1 (D2D11; Cell Signaling Technology), anti-mouse LAMP1 (1D4B; Bio-Rad), anti-LC3 (PM036; MBL), anti-LC3 (L7543; Sigma-Aldrich), anti-GFP (A-11122; Invitrogen), anti-p62 (61832; BD Pharmingen), anti-galectin-3 (M3/38; BioLegend), anti-α-tubulin (DM1A; Sigma-Aldrich), anti-CatD (ERP3057Y; ab75852), anti-CatB (D1C7Y; Cell Signaling Technology), anti-Rab10 (D36C4; Cell Signaling Technology), anti-phospho-Thr73 Rab10 (MJF-R21 [ab230261]; Abcam), anti-ATG16L1 (D6D5; CST), anti-ATG5 (A0731; Sigma-Aldrich), anti-FIP200 (17250-1-AP; Proteintech), anti-ATG13 (SAB4200100; Sigma-Aldrich), and anti-Rab29 (MJF-R30-124; Abcam). The following reagents were used at final concentrations as indicated: CQ (50–100 μM; Sigma-Aldrich), LLOMe (1 mM; Sigma-Aldrich), and zymosan (Sigma-Aldrich).

MSCs and CD4⁺ T cells from different groups were lysed, and protein was quantified as described previously. Equal amounts of protein were separated by 10% and 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing Tween 20 for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-LC3 (L7543; Sigma-Aldrich), anti-p62 (#8025; Cell Signaling Technology), anti-TGF-β1 (ab27969; Abcam), anti-CXCL8 (208-IL-010; R&D Systems), or anti-GAPDH. After washing three times, the PVDF membranes were incubated with the corresponding secondary antibodies (1:3000; Santa Cruz Biotechnology) for 1 h at room temperature. Specific antibody-antigen complexes were detected using Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (Millipore). The intensity of each band was determined using ImageJ software (version 1.49e).