Phospho erk1 2 antibody

Immunostaining was performed as described above, using phospho-ERK1/2 antibody (Thr202/Tyr204) (1:800, D13.14.4E, Cell signaling, Danvers, USA). For the western blot analysis, total tumor lysates were generated as previously described [20 (link)]. Thirty micrograms of protein per sample were separated electrophoretically in 4–15 % precast SDS polyacrylamide gels and transferred to precut nitrocellulose membranes using the Trans-Blot® turbo™ Transfer Starter System (all Bio-Rad). Chemiluminescence and colorimetric detection were performed using ChemiDoc™ MP Imaging System and horseradish peroxidase conjugated rabbit polyclonal anti-human p-ERK1/2 (Thr202/Tyr204), ERK1/2 (1:1000) detected with peroxidase-conjugated secondary anti-rabbit antibody, respectively (1:1000; Cell Signaling Technology) and HRP conjugated mouse monoclonal antibody anti-human β-Actin (S125; all 1:1000), followed by chemiluminescence solution (Clarity™ Western Chemiluminescent HRP Substrate; Bio-Rad).

HH cells were cultured in six-well plates in the presence or absence of anti-human CD147 antibody (1 µg/mL; Biolegend) or anti-human CypA antibody (0.7 µg/mL; Abcam) for 1, 3, or 6 h. After collecting proteins from HH cells, equal amounts of proteins were subjected to 4–12% NuPage Bis-Tris Gels (Invitrogen, Waltham, MA, USA) at 200 V for 20 min. The proteins were then transferred onto polyvinylidene fluoride membranes (Invitrogen) and blocked in 2% skim milk powder with 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA) in Tris-buffered saline. The membranes were probed with Akt Antibody (Cell Signaling Technology, Danvers, MA, USA), Phospho-Akt Antibody (Cell Signaling Technology), ERK1/2 Antibody (Cell Signaling Technology), Phospho-ERK1/2 Antibody (Cell Signaling Technology), p38 MAPK Antibody (Cell Signaling Technology), Phospho-p38 MAPK Antibody (Cell Signaling Technology), SAPK/JNK Antibody (Cell Signaling Technology), Phospho-SAPK/JNK Antibody (Cell Signaling Technology), or β-actin Antibody (Cell Signaling Technology) as primary antibody overnight at 4 °C, followed by incubation in secondary antibody for 30 min at room temperature. Visualization was performed by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA).

Morphine was a kind gift from the University of Sydney, Australia. Pregabalin and gabapentin were purchased from Tocris, Australia. Forskolin was provided by Ascent Scientific Ltd. Culture reagents and buffer salts were supplied by Sigma Aldrich (Castle Hill, Australia) or Thermo Fisher Scientific (Waltham, MA, USA). The CAMYEL plasmid encoding the cAMP sensor YFP-EPac-RLuc was originally from ATCC (MBA-277). Phospho-ERK1/2 antibody and anti-rabbit IgG HRP linked antibody were from Cell Signaling Technologies (Danvers, MA, USA). Antibiotics were from Invivogen (San Diego, CA, USA) and coelenterazine h was from Promega (Alexandria, Australia). The membrane potential dye was purchased from Molecular Devices, CA.

Recombinant Human FGF5 (R&D, 237-F5), anti-FGF5 antibody (Abcam, ab88118), anti-FGFR1 antibody (Abcam, ab10646), anti-FGFR2 antibody (Abcam, ab10648), CD206 antibody (R&D, AF2535), Phospho-ERK1/2 antibody (Cell Signaling, #9101), Total-ERK1/2 antibody (Cell Signaling, #9102), N-cadherin (Becton-Dickinson, 610920) and GAPDH antibody (EMD Millipore, MAB374) were used. Hoechst and species-specific secondary antibodies conjugated with Alexa Fluor 488 or 568 dyes were purchased from Invitrogen. Horseradish peroxidase (HRP) conjugated secondary antibodies for western blotting were purchased from Sigma.

Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to NC membranes, and blocked with TBST of 5% skim milk for 1 hour. The membranes were washed with TBST and then incubated with the specific primary antibody for 6 hours at 4°C. The membranes were then incubated for 1 hour at room temperature in secondary and the signal was detected by chemiluminescence (Bio rad, USA). The primary (1:1000) and secondary (1:10000) antibodies were purchased from Cell Signaling Technology, USA and listed as follows: GAPDH antibody(#2118), stat3 antibody(#12640), phospho-stat3 antibody(#98543), SAPK/JNK antibody(#9252), phospho-SAPK/JNK antibody(#4668), p65 antibody(#4764), phospho-p65 antibody(#3033), IKKα antibody(#2682), phospho-IKKα/β antibody(#2697), IκBα antibody(#4812), phospho-IκBα antibody(#2859), p38 MAPK antibody(#8690), phospho-p38 MAPK antibody(#4511), Erk1/2 antibody(#4695), phospho-Erk1/2 antibody(#4370) and HRP-linked goat anti-rabbit IgG(#7074).