Sds sample buffer

Immunoblotting was performed as previously described (17 (link)). Briefly, following cell lysis and measurement of protein concentrations, the cells were dissolved in sodium dodecyl sulfate (SDS) sample buffer (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts of protein were analyzed by SDS-PAGE on 12% polyacrylamide gels (Bio-Rad) and the proteins were electroblotted onto polyvinylidene fluoride membranes (Roche Diagnostics GmbH). The membranes were incubated in 5% non-fat dry milk in Tris-buffered saline (NaCl/Tris; Boster Biological Engineering Co., Ltd., Wuhan, China) containing 0.1% Tween-20 (Sigma-Aldrich) for 1 h at room temperature overnight at 4°C, and subsequently incubated with primary antibodies. Following incubation with a horseradish peroxidase-labeled secondary antibody, protein exposure was achieved with a molecular imager (ChemiDOC™ XRS+, Bio-Rad).

PLGA and PLGA + PC NPs were denatured by heating the NPs in SDS sample buffer (Beyotime, China) for 5–10 min at 95 ℃ and separated by electrophoresis on 10% polyacrylamide precast gels. The resulting gels were stained with Coomassie Stain (Bio-Rad) over-night and destained in methanol/water (1:3, v/v) for 12 h. Stained gels were imaged using an ImageQuant LAS4010 image analyzer (GE Healthcare).
Determination of serum protein adsorbed by PLGA NPs was carried out according to the standards bicinchoninic acid (BCA) protein assay kit. PLGA NPs were incubated with 2 mL mouse serum for 30 min, the mixture was centrifuged at 3000 rpm for 20 min, and then the supernatant was collected to determine protein content by the BCA kit. Meantime, untreated serum was the control group.

After cells treated with different cell culture medium for 48 h, cells were treated with 10 µM MG132 (MedChem Express, HY-13259) for 6 h before harvesting. Then cells were collected and whole proteins were withdrawn using a mixture of 100 µL of lysis buffer, consisted of 1 µL of protease inhibitor (100 ×), 1 µL of phosphatase inhibitor (100 ×) and 1 µL of benzenesulfonyl fluoride (100 ×). Protein samples were denatured in 10% SDS sample buffer (Beyotime Biotechnology, China) and separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) under denaturing conditions. After protein was dissociated, it was electroblotted onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA), blocked with 5% skim milk powder, and then and incubated with diluted primary antibody overnight at 4 ℃. Then the hybridization bands were incubated with horseradish peroxidase-conjugated secondary antibodies (Zhongshan, Beijing, China) at 37 ℃ for 1 h. Protein concentrations were measured using the commercially available BeyoECL Star ultra-sensitive ECL chemiluminescence kit (Beyotime Biotechnology, China). Protein expression was quantified with ImageJ software (NIH, Bethesda, MD, USA). Primary antibodies were β-actin (1:1000, TA-09, Zhongshan, Beijing, China) and P53 antibody (1:1000, 2524S, CST, USA).

Cells were washed twice in 2 mL ice-cold PBS and harvested with a rubber policeman after being lysed on ice for 20 min in lysis buffer (Beyotime, Shanghai, China). Equal amounts of total protein were solubilized by sodium dodecyl sulfate (SDS) sample buffer (Beyotime, Shanghai, China), separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Membranes were incubated with corresponding polyclonal and secondary antibodies. Signal was detected with a Super ECL Reagent substrate (Hai Gene, Harbin, China). Source and product number of antibody: LC3B (Abcam, ab48394), SQSTM1/p62 (Abcam, ab101266), Beclin 1 (Bioworld, AP0769), p-ULK1 (Affinity Biosciences, AF4387), ULK1 (Proteintech, 20986-1-AP), p-ATG13 (Bioworld, BZ40743), ATG13 (Bioworld, bs6045), ATG5 (Proteintech, 10181-2-AP), p-PI3K (CST, 4228), PI3K (CST, 4249), p-PTEN (CST, 9554), PTEN (CST, 9188), p-Akt (CST, 13038), Akt (CST, 4691), p-mTOR (Abcam, ab84400), mTOR (CST, 2972S), p-p70 S6K (CST, 9205), p70 S6K (CST, 2708), p-4E-BP1 (CST, 2855), 4E-BP1 (CST, 9644), GAPDH (Bioworld, AP0066), HRP-linked antibody (anti-rabbit IgG, CST, 7074), Goat Anti-Rabbit (Alexa Fluor®488) (Abcam, ab150077), goat anti-rabbit (Alexa Fluor®647) (Abcam, ab150079).

The antibodies used were also listed in Supplementary Table S2. As we described previously^{11 (link)}, cells were extracted for 30 min with immunoprecipitation lysis buffer (Beyotime). After centrifugation of the preparations, the concentrations of supernatants were measured with the BCA kit. Then 100 μg proteins were incubated with 14-3-3ε antibody at 4 ˚C overnight. Then the protein-antibody complex were incubated with IgA plus IgG sepharose beads (Beyotime) at 4 ˚C for another 12 h. After then, the supernatants were removed and the beads were washed for three times, resuspended in the SDS sample buffer (Beyotime), and boiled to remove protein from the beads. Then, such protein samples were analyzed by western blots.

Peritoneal macrophages were treated with 1 μg/mL LPS for 20 min. The cells were washed with PBS and proteins were extracted via treatment with RIPA buffer (Beyotime) at 4°C for 30 minutes. Cells were centrifuged at 10,000 g for 30 minutes at 4°C, allowing cell debris to be pelleted and discarded. Cellular protein (100 μg) was mixed with 1 μg of anti-Appl1 or Appl2 antibody and incubated over-night at 4°C. Then, 10 μl of protein G Plus-agarose (Santa Cruz) was added to these samples and incubated for another 4 h at 4°C. After the incubation, samples were washed three times with lysis buffer. The washed samples were re-suspended in SDS sample buffer (Beyotime) and heated at 100°C for 5 min prior to electrophoresis.