Anti igfbp2

The mice retinal tissues were subjected to milling and lysis using RIPA reagent (Beyotime, Shanghai, China) supplemented with a protease inhibitor, aiming to extract proteins. The isolated proteins were concentrated via the BCA method and then subjected to boiling at 95°C for 5 minutes. For vertical electrophoresis, 20 μg of protein was introduced into each well and separated by 12% SDS-PAGE at 110V for 1 hour. Subsequently, the proteins were transferred onto a PVDF membrane. The PVDF membrane was subsequently blocked using blocking serum for 1 hour before being incubated with primary antibodies, namely Anti-COL6A3 (1:1000; Abcam, Cambridge, MA, USA) and Anti-IGFBP2 (1:1000; Abcam, Cambridge, MA, USA), overnight at 4°C. The membrane was then exposed to a secondary antibody (1:1000; Abcam, Cambridge, USA) at room temperature for 1 hour. Finally, enhanced chemiluminescence reagent was added for protein band development, and the grayscale value of the protein bands was scanned using QUANTITY ONE software.

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.