0.45 μm pvdf membrane

Total protein was extracted by 1× SDS protein lysis buffer supplemented with protease inhibitors (MCE, Monmouth Junction, NJ, USA) and phosphatase inhibitors (MCE, Monmouth Junction, NJ, USA). Total protein was separated in 12% SDS-PAGE and transferred to 0.45 μm PVDF membranes (EMD Millipore, Burlington, MA, USA). After blocking with 5% bovine serum albumin, the membranes were incubated respectively with Rabbit-anti Cdc42 (CST, Danvers, MA, USA), JNK (CST, Danvers, MA, USA), phospho-JNK (CST, Danvers, MA, USA), p44/42 MAPK (ERK1/2) (CST, Danvers, MA, USA), phospho-p44/42 MAPK (ERK1/2) (CST, Danvers, MA, USA), p38 (CST, Danvers, MA, USA), phospho-p38 (CST, Danvers, MA, USA), p65 (CST, Danvers, MA, USA), phospho-p65 (CST, Danvers, MA, USA), HSP90 (CST, Danvers, MA, USA), β-Tubulin (CST, Danvers, MA, USA) and GAPDH primary antibodies (CST, Danvers, MA, USA), then conjugated with Goat anti-Rabbit secondary antibody (Invitrogen, Gaithersburg, MD, USA). Proteins were visualized by Odyssey CLx Infra-Red Imaging system (Odyssey CLx, Lincoln, NE, USA).

Total protein was extracted from imDCs, mDCs and Met-mDCs using RIPA buffer containing protease (phenylmethylsulfonylfluoride (PMSF)) and phosphatase inhibitors (Solarbio, Beijing, China). The protein concentration was determined by a BCA kit (Servicebio, Beijing, China). Equal amounts of denatured protein samples were separated on a 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) gel and transferred onto 0.45 μm PVDF membranes (Merck Millipore Ltd., Tullagreen, Carrigtwohill Co. Cork, Ireland). After being blocked with 5% nonfat milk, the membranes were incubated with primary antibodies against GAPDH, pFoxO3a (s253) and Foxo3a overnight at 4 °C and then with HRP-conjugated secondary antibodies for 1 h at room temperature. The signals were visualized using an enhanced chemiluminescence detection kit (Meilunbio, Dalian, China) in an imaging system (Bio-Rad, Hercules, CA, USA). The mean gray value of each band was analyzed by ImageJ software.

ESCs and macrophages were extracted by precooled RIPA lysis buffer containing 1% protease and phosphatase inhibitors (Beyotime, China). Total proteins of cell concentration were calculated using a BCA Protein Assay Kit (Beyotime). Proteins were separated by SDS-PAGE (8–12%) and transferred onto 0.45 μm PVDF membranes (Merck Millipore, USA). Then, the membranes were blocked with 5% (w/v) BSA solution for 2 h at room temperature. Next, the membranes were incubated with primary antibodies (Supplementary Table S1) at 4°C overnight. The membranes were washed with TBST solution three times and then incubated with secondary antibodies (1:10 000, Proteintech, China) for 2 h at room temperature. The signals of proteins were visualized by enhanced chemiluminescence kit (ECL, CA) and exposed with a ChemiDoc XRS system (Bio-Rad, USA). The protein expressions were normalized by GAPDH, and images were analyzed by Image Lab software (Bio-Rad).

Protein detection were performed in accordance with previous reports [20 ]. Cells were inoculated into Petri dishes (10 cm) and then treated with the same immunofluorescence procedure as those collected after 24 h of NIF administration. Collected tissue was frozen in liquid nitrogen and ground into a powder in a mortar. Lung tissue and cultured cells were homogenized in RIPA buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors (Selleckchem) and quantified using the Bradford method. Then, the proteins were separated on 10% SDS–PAGE gels (Chengdu Baihe Technology Co., Ltd.) and transferred onto 0.45 μM PVDF membranes (Merck Millipore, Billerica, MA, USA). After blocking in 5% (M/V) non-fat milk for 1 h, the membranes were incubated with antibodies overnight at 4 °C. Then, the membranes were incubated with goat anti‐rabbit/mouse IgG (ZSGB-BIO Co., Beijing, China) at a 1:3000 dilution for 60 min at 37 °C. Reactive bands were identified using an enhanced chemiluminescence kit (Merck Millipore, Billerica, MA, USA). Then, the images were analysed using ImageJ software (National Institute of Health, Bethesda, MD, USA).

Total protein was extracted from imDCs, mDCs and Met-mDCs using RIPA buffer containing protease (phenylmethylsulfonylfluoride (PMSF)) and phosphatase inhibitors (Solarbio, Beijing, China). The protein concentration was determined by a BCA kit (Servicebio, Beijing, China). Equal amounts of denatured protein samples were separated on a 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) gel and transferred onto 0.45 μm PVDF membranes (Merck Millipore Ltd., Tullagreen, Carrigtwohill Co. Cork, Ireland). After being blocked with 5% nonfat milk, the membranes were incubated with primary antibodies against GAPDH, pFoxO3a (s253) and Foxo3a overnight at 4 °C and then with HRP-conjugated secondary antibodies for 1 h at room temperature. The signals were visualized using an enhanced chemiluminescence detection kit (Meilunbio, Dalian, China) in an imaging system (Bio-Rad, Hercules, CA, USA). The mean gray value of each band was analyzed by ImageJ software.

Cellular proteins were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was determined by the BCA method. A total of 40 μg of protein for each group was separated on 12% SDS-PAGE gels and transferred to 0.45-μm PVDF membranes (EMD Millipore). Membranes were then blocked with 5% BSA and incubated with primary antibodies at 4°C overnight against HPS70 (4876, CST), CD9 (ab92726, CST), CD63 (ab193349, Abcam), RBP4 (ab233138, Abcam), RPSA (ab133645, Abcam), RPS3(9538, CST), RPS20 (ab133776, Abcam), RPS14 (ab246916, Abcam), RPL4 (ab234829, Abcam), RPL13 (ab134961, Abcam), HSPD1 (ab46798, Abcam), HSPA8 (8444, CST), P-gp (13342, CST), p-cofilin-1 (3313, CST), cofilin-1 (5175, CST), PP1 (sc-7482, Santa Cruz), PP2A (9780, CST). Anti-β-actin (ab179467, Abcam) and anti-GAPDH (2118, CST) were used as the internal control. Anti-COX IV (11967, CST) was used as loading control for mitochondrial proteins. The membranes were then incubated at 37°C for 1 h with an HRP-conjugated secondary antibody (ab97051, Abcam). Bands were visualized by chemiluminescence according to the manufacturer’s protocols.