Mouse anti p erk

Ten-micrometer sections from rat L4–L6 spinal cords were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 35 min. Endogenous peroxidase activity was quenched using 3% H₂O₂ for 30 min and then non-specific binding sites were blocked by 30 min incubation in blocking solution (Vector Laboratories, Burlingame, CA, USA) at room temperature. Subsequently, the slides were incubated overnight in goat anti-IGF2 antibody (1:100; Santa Cruz Biotechnology, Dallas, TX, USA, sc-7435) at 4 °C and then for 1 h in HRP-conjugated antibody at room temperature. The IGF2 immunoreactive signal was developed using a Cy-3 Tyramide Signal Amplification (TSA) System (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s instructions. For double staining, slides were then heated in a microwave oven to remove IGF2 antibody, as described by Toth and Mezey with minor modifications [53 (link)]. Briefly, the slides were stained using mouse anti-pERK (1:100; Santa Cruz Biotechnology, sc-7383), mouse anti-GFAP (1:100; EMD Millipore, Billerica, MA, USA, MAB3402), anti-NeuN (1:25; EMD Millipore, MAB377), and mouseanti-CD11b (1:100; Abcam, Cambridge, UK, ab75476) antibodies. Finally, all signals from double staining were developed using a fluorescein isothiocyanate TSA system.

Bovine thrombin and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). Mouse anti-p-ERK, rabbit anti-ERK, goat anti-AKT1/2, and rabbit anti-p-AKT1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-CD41 antibody was obtained from Abcam (Cambs., UK). Horseradish peroxidase- (HRP-) labeled goat anti-mouse immunoglobulin G (IgG), goat anti-rabbit IgG, and rabbit anti-goat IgG antibodies were obtained from Jackson Immuno Research (West Grove, PA, USA). R-phycoerythrin- (R-PE-) conjugated goat anti-rabbit IgG (H + L) was acquired from Proteintech (Chicago, USA). Fluorescein isothiocyanate- (FITC-) conjugated anti-CD61 antibody was purchased from eBioscience (CA, USA).

Cis-diammineplatinum dichloride (CDDP), doxorubicin hydrochloride (DXR) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO).
Monoclonal rat anti-Gal-3 antibody was isolated from the supernatant of hybridoma (catalog number: TIB-166, American Type Culture Collection, Manassas, VA); customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Carlsbad, CA); mouse anti-V5 was purchased from Invitrogen; polyclonal rabbit anti-Bax, mouse anti-Bcl-xl, mouse anti-p-ERK, mouse ERK1/2 and rabbit anti-p-Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); mouse anti-β-actin was purchased from Sigma-Aldrich; monoclonal mouse anti-Bax was purchased from BD Biosciences (San Diego, CA); mouse anti-p21, rabbit anti-Bcl2, rabbit anti-PARP (9542L), rabbit anti-cleaved caspase-3, rabbit anti-Akt and mouse anti-caspase-8 were purchased from Cell Signaling Technology (Beverly, MA); purified rabbit IgG was purchased from ZYMED (San Francisco, CA);

HepG2 cells were harvested and lysed in a RIPA buffer containing leupeptin, 1 mM PMSF, Na₃VO₄, and 0.1% protease inhibitor cocktails (Sigma, St. Louis, MO, USA). After 30-min incubation on ice, the cells were centrifuged at 13,000 rpm for 20 min at 4°C. The supernatant was mixed with a 5× sample buffer and then boiled for 10 min. Equal amounts of protein cell lysates were loaded onto an SDS-polyacrylamide gel for electrophoresis. PVDF membrane (Millipore, Billerica, MA, USA) was used to transfer proteins from the gel. Mouse anti-SREBP-2 (Santa Cruz Biotech., Santa Cruz, CA, USA), rabbit anti-LDLR (BioVision, Milpitas, CA, USA), rabbit anti-p-JNK (Merck Millipore, Billerica, MA, USA), rabbit anti-p-p38 (Merck Millipore, Billerica, MA, USA), mouse anti-p-ERK (Santa Cruz Biotech., Santa Cruz, CA, USA), and rabbit anti-GAPDH (Signalway antibody, Pearland, TX, USA) antibodies were used to detect the proteins.