Rabbit anti erk1 2 antibody

For Western blot analysis, cells were lysed with Triton lysis buffer (TLB; 20 mM Tris-HCl, pH 7.4; 137 mM NaCl; 10% Glycerol; 1% Triton X-100; 2 mM EDTA; 50 mM sodium glycerophosphate; 20 mM sodium pyrophosphate; 5 μg/mL aprotinin; 5 μg/mL leupeptin; 1 mM sodium vanadate; 0.2 mM Pefabloc and 5 mM benzamidine) for 30 min at 4 °C. Cell lysates were centrifuged, and 5× Laemmli buffer (10% SDS, 50% glycerol, 25% 2-mercaptoethanol, 0.02% bromophenol blue, 312 mM Tris, 6.8 pH) was added (1:5 dilution) to the supernatant. The mixture was boiled at 95 °C for 10 min. Samples were subjected to SDS-PAGE and Western blot analysis. SARS-CoV-2 spike protein was detected using a rabbit polyclonal anti-SARS-CoV-2 spike S2 antibody (#40590-T62, Sino Biological, Hong Kong, China). Equal protein load was verified using a mouse monoclonal anti-ERK2 antibody (sc-1647, Santa Cruz, CA, USA) or rabbit anti-ERK1/2 antibody (#4695, Cell Signaling Technology, Danvers, MA, USA). Quantification was performed using Fiji (Image J. JS v0.5.7; https://ij.imjoy.io). Samples were normalised using the loading control.

The primary antibodies used for p-ERK1/2, ERK1/2, or β-tubulin detection were: rabbit anti-phospho-ERK1/2(Thr²⁰²/Tyr²⁰⁴) antibody (1:2000; Cell Signaling Technology), rabbit anti-ERK1/2 antibody (1:2000; Cell Signaling Technology), and beta-tubulin rabbit monoclonal antibody (1:2000; Beyotime). To examine the A. japonicus kisspeptin precursor or AjKissR1 in various tissues of sea cucumber, AjKiss1b-10 or a peptide corresponding to amino acids Ser¹⁵⁰~Trp¹⁷⁴ of AjKissR1, the second intracellular loop, was synthesized and injected into two rabbits, respectively. The polyclonal antibodies, rabbit anti-AjKiss1b-10 (1:1000) was prepared by ChinaPeptides, and anti-AjKissR1 (1:1000) was prepared by Wuhan Transduction Bio. The secondary antibodies used were HRP-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-rabbit IgG, and Cy3-conjugated goat anti-rabbit IgG (Beyotime).

After the cell lysate was extracted with cell lysis buffer (20 mM Tris-HCl pH 7.7, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) from HCS-2/8 and RCS cells, the concentration of proteins in the cell lysates were determined by the BCA protein assay kit (Thermo Fisher Scientific) using a bovine serum albumin (BSA) standard curve. Then, Western blot analysis was performed as described previously [31 (link)]. The following primary antibodies were used: mouse anti-AT₁ receptor antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-CCN2 antibody (Abcam), rabbit anti-MMP9 antibody (Triple Point Biologics, Inc., Forest Grove, OR, USA), rabbit anti-ERK1/2 antibody (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-phospho-ERK1/2 antibody (Promega, Madison, WI, USA), and mouse anti-β-actin antibody (Sigma-Aldrich). The following secondary antibodies were used: horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (SeraCare Life Sciences, Inc., Milford, MA, USA) or HRP-conjugated anti-mouse IgG antibody (Rockland, Limerick, PA, USA).

The same amount of protein was loaded onto an SDS-PAGE gel, transferred to a nitrocellulose (NC) membrane, and blocked with 50 g/L non-fat milk for 1 h. The membrane was then incubated with the following antibodies at 4°C: rabbit anti-CD147 antibody (MAB972-100, R@D, 1:3,000) and rabbit anti-ERK1/2 antibody (Cell Signaling Technology, 1:5,000), rabbit anti-phospho-ERK1/2 antibody (Cell Signaling Technology, 1:5,000), rabbit anti-Sp1 antibody (sc-14027, Santa Cruz Biotechnology, 1:2,000), rabbit anti-MMP2 antibody (ab86607, abcam, 1:3,000) rabbit anti-MMP9 antibody (ab236494, abcam, 1:2,000), and mouse anti-β-Actin (sc-8432; Santa Cruz Biotechnology,1:5,000). The NC membrane was washed with TBST 3 times for 10 min each time and was then incubated with a secondary antibody at room temperature for 1.5 h, followed by 3 washes in TBST. The membrane was then treated with enhanced chemiluminescence (ECL) reagent. The membrane was scanned and the absorbance (A) values of the protein bands were analysed.