Ripa lysate

Total exosome protein was extracted using RIPA lysate (Sigma-Aldrich, USA) and protease inhibitor PMSF (Beyotime, China), Western blot analysis was performed on a 10% SDS-PAGE gel, and Page RulerTM Prestained Protein Ladder (MBI Fermentas, Lithuania) was used as the upper sample marker. The primary antibody was anti-CD63 (1:2000, Genechem, China) and TSG 101 rabbit polyclonal antibody (Ab) (1:2000, Genechem, China). Samples were incubated at 4°C overnight before a further 2 hour incubation with goat anti-rabbit secondary antibody (1:10000; Bioword, USA). The SuperSignal West Dura Extended Duration Substrate Kit (Thermo Scientific, China) was used to visualize bound proteins.

Total protein in skin flap was collected by RIPA lysate (R0278, Sigma-Aldrich, Missouri, USA) mixed with protease inhibitor (S8830, Sigma-Aldrich, Missouri, USA) in order to determine the protein levels of RIP1, RIP3, MLKL, PGAM5, Drp1, MKP-1, p-ERK1, p-ERK2, ERK1 and ERK2. The concentration of total protein was evaluated by BCA kit (Sigma-Aldrich, Missouri, USA) in the first place to estimate the loading volume of samples onto the SDS-PAGE for separation. Parameter for electrophoresis was 100 V for 2 h. PVDF membrane was used for the transferring of protein separated. Following the blocking of membrane for 60 min, primary antibodies against RIP1 (CST, #3493, 78 kD, 1:1000), RIP3 (Abcam, ab56164, 57 kD, 1:1000), MLKL (CST, #37705, 54 kD, 1:1000), PGAM5 (Abcam, ab126534, 32 kD, 1:1000), Drp1 (Abcam, ab56788, 82 kD, 1:1000), MKP-1 (CST, #3493,78 kD, 1:1000), p-ERK1/2 (Abcam, ab50011,42–44 kD, 1:1000), ERK1/2 (Abcam, ab17942, 42–44 kD), GAPDH (Abcam, ab8245, 36kD, 1:2000) were incubated on the membrane at 4 °C overnight. Next day, after the washing of membrane for 3 times, primary antibodies were probed by Goat anti-rabbit secondary antibody (Abcam, ab205718, 1:2000) and developed by ECL (#6883, SignalFire™ ECL Reagent) after washing by PBST (PBS with 0.1% Tween).

The total cellular or liver tissue protein was obtained by lysing the cells with RIPA lysate (Sigma, V900854), and the protein concentration was determined using a BCA protein concentration assay kit (Sigma, FP0010). Equal amounts of protein were electrophoresed on a 10% Bis-Tris gel at 120 V for 1 h, the protein was transferred to the PVDF membrane at 350 mA for 70 minutes, and the PVDF membrane was blocked with 5% BSA in TBST buffer for 1 h. The primary antibody was incubated by gentle shaking at 4°C overnight, and the secondary antibody was incubated for 1 h at room temperature. ECL hypersensitive luminescent solution (Thermo, 32132) was used for color reaction, and gray scale was detected by image laboratory software (Bio-Rad) to quantitatively analyze protein expression. The antibodies used included HNF1α antibody (Abcam, ab96777), IRS-1 antibody (CST, #2382), phospho-IRS-1 antibody (CST, #2385), AKT antibody (CST, #9272), phospho-Akt antibody (CST, #4060), SOCS3 antibody (CST, #2932), STAT3 antibody (CST, #9139), phospho-STAT3 (CST, #9134), SREBP1 antibody (Abcam, ab191857), and PPARα antibody (Abcam, ab8934). The GAPDH protein antibody (Abcam, ab8245) was selected as an internal reference.

The total protein was extracted using RIPA lysate (#R0278, Sigma) according to the manufacturer's instructions. The BCA protein assay kit (Beyotime, Shanghai, China) was used to measure the protein concentration according to the manufacturer's instructions. An equal amount of protein was loaded onto sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and immunoblotting was performed as described previously [24 (link)]. Primary antibodies against HO-1 (1 : 1000), Nrf2 (1 : 1000), Bax (1 : 1000), Bcl-2 (1 : 1000), NF-κB p65 (1 : 1000), c-PARP (1 : 1000), GAPDH (1 : 2000), phosphor-NF-κB p65 (1 : 1000), p-IκB-α (1 : 1000), and IκB-α (1 : 1000) were used. HRP-conjugated secondary antibodies were used to detect fluorescence using BeyoECL Plus as described previously [24 (link)].

The skin flap specimens were collected and placed in the RIPA lysate (Sigma, USA) containing protease inhibitor. After lysis on ice for 30 min, the filtrate was collected and stored under − 20℃. The BCA Protein Quantification Kit (Vazyme, China) was used to measure the protein concentration. After electrophoretic separation of equivalent proteins on 10% SDS-PAGE gel, the proteins were blotted to the PVDF membrane. After transferring membrane, the specimen was mounted at room temperature for 2 h, then it was added with primary antibody for incubation under 4℃ overnight, in the next step, secondary antibody was added for incubation for 2 h, and finally, the chromogenic agent was added for exposure.

The cells or exosomes were lysed with RIPA lysate (Sigma) to obtain proteins. The BCA Assay kit (Solarbio, Beijing, China) was used to determine the protein concentration. An equal amount of protein was added onto 10% SDS-PAGE gels, and the protein was then transferred onto polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The blots were then incubated with primary antibodies, Anti-PI3Kγ antibody (1 : 1,000, ab32089, Abcam), Anti-CD163 antibody (1 : 1,000, ab213612, Abcam), Anti-CD206 antibody (1 : 1,000, sc-58986, Santa Cruz Biotechnology), Anti-PTEN antibody (1 : 1,000, ab267787, Abcam), Anti-AKT antibody (1 : 1,000, ab213612, Abcam), Anti-pAKT antibody (1 : 1,000, ab38449, Abcam), Anti-Vimentin antibody (1 : 1,000, ab20346, Abcam), Anti-α-SMA antibody (1 : 1,000, ab108424, Abcam), Anti-IL-10 antibody (0.5 µg/ml, ab134742, Abcam), Anti-CD63 antibody (1 µg/ml, ab38418, Abcam), Anti-CD61 antibody (1 : 1,000, ab119992, Abcam), Anti-HSP70 antibody (1 : 1,000, ab2787, Abcam), and anti-Actin (1 µg/ml, ab8286, Abcam) overnight at 4°C. The horseradish peroxidase-conjugated secondary antibody was then added to incubate the blots at room temperature for 2 h. At last, the blots were visualized with the Novex™ chemiluminescent substrate reagent kit (Waltham, MA, USA).

The RIPA lysate (Sigma, America) and PMSF (Sigma, America) were used to lyse and extract proteins at a ratio of 100:1. The concentration of the centrifuged supernatant was measured using a BCA kit (Beyotime, China), and then the protein fluid was mixed with loading buffer (Solarbio, Beijing, China) and denatured at 100 °C for 5 min. The extracted lysates were separated by 10% SDS-PAGE and transferred onto PVDF membrane. Next, the membranes were incubated with primary antibody (β-actin from Abcam, America; ARK5 form Cell Signaling Technology and Santa Cruz Biotechnology, America) overnight and then were incubated with secondary antibody (anti-rabbit IgG/anti-mouse IgG, ZSGB-Bio, China) for 1.5 h. Finally, protein expression can be observed by chemiluminescence and analyzed using Image lab.