P erk antibody

The CECs scraped off the corneas that served as controls during wounding and at the end of experiment were collected and frozen immediately in liquid nitrogen and stored at an Eppendorf tube at −80°C. For western blot, human and mouse CECs were lysed with radioimmunoprecipitation assay buffer. The lysates were centrifuged to obtain the supernatant. Protein concentration was determined by bicinchoninic acid (BCA) assay. The protein samples were separated by SDS-PAGE and electrically transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 3% BSA and subsequently incubated with primary and secondary antibodies. Signals were visualized using Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate (#34580; Thermo Fisher Scientific, Waltham, MA, USA) using an Invitrogen iBright Imaging System (Thermo Fisher Scientific). Antibodies to phospho-Akt (p-Akt, #9271, 1:500 dilution), p-EGFR (#4470, 1:500 dilution), and p-SMAD2/SMAD3 (#8828, 1:500 dilution) were obtained from Cell Signaling Technology (Danvers, MA, USA); p-ERK antibody (#sc-7383, 1:1000 dilution) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and anti-β-actin antibody (#A1978, 1:10000 dilution), which served as the loading control, was obtained from Sigma-Aldrich (St. Louis, MO, USA.

ERK1/2 activity was assayed by immunoblotting as previously described (Gonzalez de Valdivia et al., 2017 (link)) using phosphorylated ERK1/2 (pERK) antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:1000) for ERK1/2 phosphorylation and ERK1/2 (ERK) antibody (Santa Cruz Biotechnology; 1:1000) for total ERK1/2. Briefly, cells were grown to confluency in 60-mm dishes in phenol red–free DMEM with 10% FBS, washed, incubated without serum for 1 hour, and then incubated without or with vehicle (DMSO) or drug for different times. The cells were then washed, lysed, and subjected to immunoblotting, and immunoreactive bands were visualized as described below. The combined band densities of ERK1 and ERK2 were quantified using ImageJ software, and ERK1/2 activity was expressed as the ratio between the combined pERK band densities and the combined ERK band densities for each condition.