Catalog no 2118s

The animals were euthanized using CO₂ asphyxiation and the mouse conjunctival tissues were harvested. The conjunctiva sample underwent cold PBS washing followed by incubation in lysis buffer containing phosphatase and protease inhibitors. Tissue lysates were sonicated and homogenized, then centrifuged to obtain supernatants for protein quantification. Electrophoresis on SDS gels, protein transfer to PVDF membranes, and blocking with BSA in TBST buffer were conducted. After that, membranes were incubated overnight at 4 °C with specific rabbit polyclonal antibodies against phosphorylated NF-kB (pNF-kB; 1:1000, Catalog No. 3033S; Cell Signaling Technology, Inc.) and GAPDH (1:10,000, Catalog No. 2118S; Cell Signaling Technology, Inc.). The membranes were washed and incubated with an HRP-linked secondary antibody, and the bands were visualized with a chemiluminescence system (WBKLS0100; MilliporeSigma, Inc., Burlington, MA, USA). The protein expression levels were normalized to GAPDH in the same samples.

Western blotting was performed according to standard procedures. In brief, the proteins were extracted from individual cells in RIPA lysis buffer (Catalog no. 822721, MP Biomedicals) supplemented with protease inhibitor (Catalog no. A32953, Thermo Scientific). Protein concentration was determined using a BCA protein assay kit (Catalog no. A23225, Thermo Scientific), and whole lysates were denatured at 95 °C in 4X Sample buffer and separated on SDS–polyacrylamide gels. The separated proteins were then transferred to PVDF membrane and blocked in 10% BSA, followed by primary antibody incubation in 1% BSA overnight at 4 °C and secondary antibody incubation in 1% BSA, and then imaging of the membranes was performed. Primary antibodies were IGF-1R (Catalog no. SAB4300359, Sigma-Aldrich, St. Louis, MO, USA), ERK1/2 (Catalog no. 4696S, Cell Signaling Technology, Danvers, MA, USA), Phosphorylated ERK1/2 (pERK1/2) (Catalog no. 4370S, Cell Signaling Technology), IQGAP1 (Catalog no. 20648, Cell Signaling Technology), and GAPDH (Catalog no.2118S, Cell Signaling Technology). Secondary antibodies used were HRP-conjugated anti-rabbit (Catalog no. Ab205718, Abcam, Cambridge, UK) and anti-mouse (Catalog no. 31430, Invitrogen, Waltham, MA, USA). Densitometry analyses of the bands were performed with ImageJ software (Version 1.53k).