Erk antibody

Western blotting was performed as described previously [13 (link)]. The following antibodies were used: CCN1 antibody (Abcam), β-actin antibody (Santa Cruz Biotechnology), Bcl-xL antibody (Cell Signaling Technology), c-Myc antibody (Cell Signaling Technology), Bax antibody (Santa Cruz Biotechnology), MEK antibody (Cell Signaling Technology), phospho-MEK antibody (Ser217/221, Cell Signaling Technology), ERK antibody (Cell Signaling Technology), phospho-ERK antibody (Thr202/Tyr204, Cell Signaling Technology), β-catenin antibody (Cell Signaling Technology), phospho-β-catenin antibody (Ser33/Ser37/Thr41, Cell Signaling Technology), and Survivin antibody (Cell Signaling Technology).
Gel electrophoresis and transfer as well as the chemiluminescence detection were conducted using the Bio-Rad Laboratories system.
Nuclear protein was extracted using a nuclear protein extraction kit (Beyotime, China), and an anti-Histone H1 antibody (Santa Cruz Biotechnology) was used as the loading control.

AG1478, and geldanamycin were purchased from R&D Systems (Minneapolis, USA). Phosphatidylinositol 4-phosphate (PI4P) and Carrier 3 were purchased from Echelon Biosciences (Utah, USA). Human HSP90AB1 plasmid was kindly provided by Professor Fei Sun (Institute of Biophysics, Chinese Academy of Sciences, China) and was ligated into pcDNA3.1 (Invitrogen, Paisley, UK) vector for expression. Antibodies to PRDX2, MTA2, FASN and HSPD1 were purchased from Abcam (Cambridge, UK). Rabbit polyclonal PI4KIIα antibody was a kind gift from Pietro De Camilli (Yale University, HHMI) (Guo et al., 2003 (link)). Antibodies to HER-2, p-HER-2 (Tyr1248), EGFR and β-actin were purchased from Santa Cruz (Texas, USA). AKT antibody, p-AKT antibody, ERK antibody and p-ERK antibody were from Cell Signaling Technology (Herts, UK). SILAC DMEM was purchased from Thermo Fisher Scientific (New Hampshire, USA). [¹²C₆,¹⁴N₂]-Lys, [¹²C₆,¹⁴N₄]-Arg, [¹³C₆,¹⁵N₂]-Lys and [¹³C₆,¹⁵N₄]-Arg were purchased from Cambridge Isotope Laboratories (Massachusetts, USA). Other reagents were purchased from Sigma (Dorset, UK) unless otherwise stated.

The analysis of the activation of ERK was carried out as previously described [22 (link),23 (link),54 (link)]. GPR55-expressing HEK293 cells were incubated with LPI (final concentration, 1 µM in 0.02 % (v/v) DMSO as vehicle) in DMEM containing 5 mM HEPES–NaOH (pH 7.4) and 0.1% BSA in 35 mm dishes at 37 °C for 5 min. In some cases, GPR55-expressing HEK293 cells were pretreated with U-0126 (20 µg/mL, 1 h), Y-27632 (20 µM, 1 h), wortmannin (500 nM, 1 h), herbimycin A (20 µM, 1 h), calphostin C (20 µM, 1 h), or U-73122 (20 µM, 1 h) before LPI challenge. Following incubation, cells were harvested and washed with ice-cold Tyrode’s solution containing 5 mM HEPES–NaOH (pH 7.4). The activation of ERK was estimated via a Western blot analysis using a specific antibody for phospho-ERK (Cell Signaling Technology, MA, USA). The amount of ERK was also estimated via Western blotting using the ERK antibody (Cell Signaling Technology, MA, USA). Each signal was visualized after the blot had been incubated with the HRP-conjugated second antibody and detected using an ATTO imager with ECL reagent. Band intensity was quantified using ImageJ, and the ratio of phospho-ERK to total ERK was calculated. Data were expressed as fold stimulation (compared with vehicle alone or time 0).