Novex ecl kit

Sample preparation and immunoblotting were performed as previously described [23] (link). Membranes were incubated with primary antibodies (anti-SOD1–ab13498; anti-SOD2–ab16956; anti-catalase- ab76024; anti-GPx1–ab108427; anti-VDAC1–ab154856; anti-TFAM–ab155240; anti-PGC-1α–ab77210; anti-PGC-1β–ab176328; anti-OPA1–ab119685; anti-MFN1–ab57602; anti-MFN2–ab56889; anti-DRP1–ab56788; anti-LC3A–ab52628; anti-HK1–ab55144, all–Abcam, USA; anti-beta-actin–MA5-15739, anti-Bcl-2-13-8800, Invitrogen, USA) overnight at 4 °C with gentle shaking. After washing, the membranes were incubated with peroxidase-conjugated secondary antibodies for 1 h at room temperature. Target proteins were detected using Novex ECL Kit (Invitrogen, USA) in ChemiDoc station (Biorad, USA). Optical densities of the protein bands were measured using ImageLab Software. Protein content was normalised on β-actin.

For Western-Blot sampling, sorted cells from 5 animals were pooled. The cell pellet was lysed with 50 μL of ice-cold RIPA buffer. Thereafter, Laemmli Sample Buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was added with heating at 95 °C. The proteins were separated by 10%–12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a membrane as described previously [57 (link)]. After blocking membranes were stained overnight with antibodies to Arg1, iNOs, CCR7, IL6, IL10, FXYD2, ALOX12, or GAPDH (anti-Arginase I: sc-18351, 1:200; anti-NOS2:sc-651, anti-GAPDH:sc-25778, Santa Cruz, USA, anti-CCR7:ab32527, anti-IL6:ab7737, anti-IL10:ab9969, Abcam, UK), anti-FXYD2:PA5-75640, anti-ALOX12:PA5-78760, ThermoScientific, Waltham, MA, USA), washed thrice, and stained with HRP-conjugated antibodies (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Bands were imaged with a Novex ECL Kit (Invitrogen, Carlsbad, CA, USA) in a ChemiDoc™ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Densitometry analysis was carried out using ImageLab Software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH was used as a reference protein. Uncropped membranes after blotting are available in Supplementary Materials.

The total protein of A549 cells, A549/DDP cells, A549-exo, and A549/DDP-exo were extracted using RIPA lysate (89901; Thermo Scientific, USA) and Total exosomal RNA& Protein isolation kit (4478545; Invitrogen), and the total protein concentration of each sample was detected. An equal amount of total protein was taken to perform electrophoretic separation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, proteins in SDS-PAGE were transferred to polyvinylidene fluoride (PVDF) membranes using the wet transfer method. PVDF membranes were closed with 5% bovine serum protein (ST023-50 g; Beyotime, China) for 1 h. PVDF membrane was then added with primary and secondary antibodies of target proteins. The antibody information is shown in Supplementary Table 1. PVDF membranes were color developed using Novex™ ECL Kit (WP20005; Invitrogen), and images were collected using an Axygen® Gel Imaging System (GD-1000; Corning, USA).

The tissue was homogenized in protein solubilization buffer (MicroRotofor Lysis Kit, Bio-Rad) with tributylphosphine and cOmplete protease inhibitor cocktail (Roche) and centrifuged at 14,000 g for 30 min. The supernatant was mixed with sample loading buffer, heated at 65°C for 5 min and stored at −20°C. Protein separation was carried out in 10.0%–12.5% PAAG. The transfer to PVDF membranes (Bio-Rad) was performed in a Trans-Blot Turbo Transfer System (Bio-Rad) at standard settings. The membrane was incubated in EveryBlot Blocking Buffer (Bio-Rad) for 15 min at room temperature and stained with protein-specific antibodies overnight at 4°C, washed, and incubated with secondary HRP-conjugated antibodies for 1 h at room temperature. Chemiluminescence was visualized using Novex ECL kit (Invitrogen, United States) in a ChemiDoc Imaging System (Bio-Rad). Chemiluminescence intensity was analyzed using ImageLab software with GAPDH as normalization reference. The uncropped WB membranes are shown in the Supplementary Figure S2.

The cells were lysed in 2× lysis buffer containing 200 mM Tris-HCl, protease inhibitor cocktail (Roche, Pleasanton, CA, USA), 400 mM β-mercaptoethanol (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 4% sodium dodecyl sulfate (SDS; Serva), 0.01% bromophenol blue, and 40% glycerol (PanReac, Barcelona, Spain) and incubated at 95 °C for 1 min. Proteins were separated using 10% or 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred from the gel to PVDF membranes by a semi-wet approach using Trans-Blot^® Turbo™ RTA Mini LF PVDF TransferKit (Bio-Rad Laboratories, Inc.). Membranes were blocked with 5% milk on tris-buffered saline containing 0.1% Tween (TTBS) for 1 h at room temperature, then stained overnight with primary antibodies against GAPDH, GRP78, LC3B, involucrin, filaggrin, and subsequently with HRP-conjugated secondary antibodies (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Target proteins were visualized by Novex ECL Kit (Invitrogen, Waltham, MA, USA) in the ChemiDoc™ visualization system (Bio-Rad Laboratories, Inc.). Optical density of the protein bands was determined using ImageLab Software (Bio-Rad Laboratories, Inc.).