Anti β actin

Primary mouse anti-Oct4, anti-Sox2, and anti-Klf4 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit anti-mono-H3K27 from Abcam; rabbit anti-mono-,di-,tri-H3K4, rabbit anti-di-,tri-H3K27, and rabbit anti-Nanog from Cell Signaling Technology (Danvers, MA, USA); rabbit anti-Nestin from Sigma (Sigma, St. Louis, MO, USA); and anti-β-tubulin and anti-β-actin from Beyotime Institute of Biotechnology (Jiangsu, China). miRNA inhibitors were purchased from Biomics Biotechnologies (Jiangsu, China).

Briefly, whole cell was lysed with radio‐immunoprecipitation assay buffer (Beyotime, Shanghai, China) and separated using 10% SDS/PAGE [14]. Proteins were transferred and the membranes (Merck Millipore, Carrigtwohill, Ireland) were blocked at room temperature. Then, the anti‐PD‐L1 (dilution 1 : 2000; Abcam, Cambridge, MA, USA) or anti‐β‐actin (dilution 1 : 2000; Beyotime) antibodies were added to bind to the target proteins. Finally, horseradish peroxidase ‐conjugated secondary antibodies were added and the signals were visualized using an ECL Kit (Beyotime).

Proteins in cells and tissues were extracted with RIPA lysis buffer (Thermo Fisher). Serum proteins were extracted with Serum Protein Extraction Kit (Qcheng Bio, China). Western blot assays were performed according to details previously reported [23 (link)]. the immuno-complexes were detected with ECL Western Blotting Substrate (Thermo Fisher), visualized with BIO-RAD (BIO-RAD Gel Doc XR+, USA). The following antibodies were used (11000): anti-β-actin (Beyotime, AF0003); anti-α-tubulin (Beyotime, AF0001); anti-GAPDH (Beyotime, AF0006); anti-HSP90 (Proteintech, 60,318–1-Ig); anti-HUR (Proteintech, 11,910–1-AP); anti-EIF2S1 (Proteintech, 11,170–1-AP); anti-VEGF (Proteintech, 19,003–1-AP); anti-Calnexin (Abcam, ab92573); anti-CD63 (Abcam, ab134045); anti-TSG101 (Abcam, ab125011); anti-CD81 (Proteintech, 66,866–1-Ig); anti-STUB1 (Proteintech, 55,430–1-AP).
IHC, IF and IP was performed as previously described [24 (link)]. IHC was performed with antibodies against HUR and VEGF (Proteintech, 1:200). IF was performed with antibody against CD31 (Proteintech, 11,265–1-AP, 1:200). The images were scanned by Pannoramic SCAN (3DHistech, Hungary). IP was performed with anti-HSP90 antibody (Proteintech, 1:200) and appropriate control IgG (Merck Millipore), and the immunoprecipitate was then collected by centrifugation and analysed by SDS-PAGE.

Total protein of cell line lysate (including control and 0.5 μg/mL of MWCNTs groups) was collected, and then separated on SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The antibodies used were anti-CDK4 (Cell Signaling Technology, 1:1000, Danvers, MA, USA), anti-Cyclin D1 (Cell Signaling Technology, 1:1000, Danvers, MA, USA), anti-Cyclin D3 (Cell Signaling Technology, 1:1000, Danvers, MA, USA), anti-P₅₃ (Cell Signaling Technology, 1:1000, Danvers, MA, USA), anti-β-actin (Beyotime, 1:1000, China). The immune complexes were detected by enhanced chemiluminescence (Millipore, Billerica, MA, USA). Blots were quantified by densitometry and normalized by use of β-actin to correct for differences in loading of the proteins. For densitometric analyses, the band was quantified using Image Lab software (BioRad laboratories, Hercules, CA, USA). Each experiment was performed at least twice.

Cells were lysed using RIPA buffer and total protein was extracted from the lysate. Total protein concentration was determined using a bicinchoninic acid assay kit (KeyGen, Nanjing, China). Protein samples were separated by electrophoresis on a 13% sodium dodecyl sulfate–polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk at 25 °C for 1 h, the membrane was incubated with the following primary antibodies: anti-Bcl-2 (1:1000; Abcam, USA), anti-Bax (1:1000; Abcam), anti-P-Iκnt (1:1000; Beyotime, China), anti-Iκnt (1:1000; Beyotime, China), anti-P-p65 (1:1000; Abcam), anti-p65 (1:1000; Abcam), and anti-β-actin (1:500; Beyotime, Hangzhou, China) overnight at 4 °C. After washing with Tris-buffered saline containing 0.1% Tween 20 (TBST), the membrane was incubated with horseradish peroxidase-labeled secondary antibody IgG (1:1000; Abcam, USA) for 1 h at 37 °C. Blots were visualized with an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK) and quantified using the Image Pro-Plus 6.0 analysis system (Media Cybernetics, Rockville, MD, USA). Band density values were normalized to β-actin.