Anti p igf 1r

MSCs were plated in six-well plates at a density of 2 × 10⁵ cells/well for 24 h and treated with exosomes at a concentration of 200 µg for 48 h. After removing the medium, cells were lysed in a buffer (P0013 and 1 mM phenylmethylsulfonyl fluoride (PMSF), Beyotime Institute of Biotechnology, Shanghai, China) for 30 min at 4°C. Exosome sample preparation was similar as before. The supernatant was harvested by centrifuging at 12,000 g for 5 min at 4°C. A bicinchoninic acid (BCA) kit was used to measure protein concentrations. The proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 1 h, detected for anti-HSP70 (EXOAB-Hsp70A-1, System Biosciences, USA), anti-CD63 (EXOAB-CD63A-1, System Biosciences, USA), anti-TSG101 (28283-1-AP, Proteintech, USA), anti-IGFBP2 (11065-3-AP, Proteintech, USA), anti-IGF-2 (ab177467, Abcam, UK), anti-IGF-1R (9750T, Cell Signaling Technology, USA), and anti-p-IGF-1R (Tyr1135, 3918T, Cell Signaling Technology, USA) at 4°C overnight, and then incubated with anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (SA00001-2, Proteintech, USA) for 1 h at room temperature. The Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was used to scan and analyze the images.

Cells were washed with PBS and lysed on ice for 5 min in NP40 buffer (50 mM Tris pH 7.5, 1% NP40, 150 mM NaCl, 10% Glycerol, 1 mM EDTA) supplemented with protease and phosphatase inhibitors (Roche). Lysates were centrifuged at 15,000 rpm for 15 min and the protein concentration was quantified using BCA (Pierce). Chromatin fractionation performed as described^{8 (link)}. All lysates were freshly prepared and supplemented with Laemmli loading buffer with subsequent boiling for immunoblotting. The primary antibodies used for immunoblotting were anti-SIRT6 (1:1000, CST, #12486), anti-IGFBP2 (1:1000, Abcam, ab109284), anti-H3K4me3 (1:2500, Abcam, ab1012), anti-H3K9ac (1:1000, Abcam, Ab10812), anti-H3K27ac (1:1000, Abcam, ab4729), anti-H3K56ac (1:1000, Abcam, ab76307), anti-p-IGF-1R (1:200, Cell Signaling, #3918), anti-IGF-1R (1:1000, Cell Signaling, #3027), anti-p-AKT (1:1000, Cell Signaling, #4060), anti-AKT (1:1000, Cell Signaling, #9272), anti-p-MEK (1:1000, Cell Signaling, #9121), anti-MEK (1:1000, Cell Signaling, #2352), anti-p-ERK (1:1000, Cell Signaling, #4370), anti-ERK (1:1000, Cell Signaling, #9107), anti-KAT1 (1:1000, Santa-Cruz, sc-390562), anti-KAT2B (1:1000, Santa-Cruz, sc-13124), anti-GAPDH (1:1000, Santa-Cruz, sc-32233), anti-b-Actin (1:1000, Sigma, A5441), and anti-Vinculin (1:1000, Sigma, V9131). Uncropped western blots can be found in Supplementary Fig. 12.