Rabbit anti gapdh antibody

Lung cancer cells were centrifuged at 1500 rpm for 5 min at 4 °C and washed with cold phosphate-buffered saline and kept on ice in RIPA lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA). Lysates were centrifuged at 12,000 rpm for 10 min at 4 °C and the supernatants were kept for Western blot analysis. Equal amounts of sample were applied to an SDS-PAGE gel, then the gel was transferred into a polyvinylidene fluoride (PVDF) membrane. The transferred membrane was blocked with 10% non-fat milk, then incubated with the primary antibodies, rabbit anti-GAPDH antibody (1:2500, Abcam, USA) and rabbit anti-ASS1 antibody (1:20,000, Abcam, USA). After the incubation, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (1:5000, Abcam, USA); then, signals were detected by a chemiluminescent detection kit (Pierce, Rockford, IL, USA). The relative ASS expression level was estimated by ImageJ.

Protein extracts were prepared, separated, and transferred to membranes as previously described [66 (link)]. Membranes were blocked in 5 % nonfat dry milk (w/v) in Tris-buffered saline (pH 7.0) containing 0.1 % Triton-100 (TBS-T) for 1 h at room temperature. Membranes were incubated at 4 °C overnight with the following: 1:2,500 rabbit anti-phospho-p38 MAPK antibody (Cayman Chemical), 1:2,000 rabbit anti-phospho-MK2 antibody (Abgent), or with 1:10,000 rabbit anti-GAPDH antibody (Abcam) in 5 % non-fat dry milk in TBS-T. Anti-phospho-p38 MAPK antibody and anti-phospho-MK2 antibody were raised against mammalian proteins; peptide sequences used to produce these monoclonal antibodies have greater than 90 % identity to their counterparts in A. stephensi. Membranes were washed three times for 5 min each in 1X TBS-T and incubated with a 1:20,000 dilution of HRP-conjugated goat anti-rabbit (Fab’)2 fragment (Cell Signaling Technology) at 4 °C overnight. To reveal antibody-bound proteins, membranes were incubated with SuperSignal West Dura chemiluminescent reagent for 5 min and visualized using the Kodak Image Station 4000MM Pro and Carestream Molecular Imaging software (Carestream Health). Levels of phospho-proteins in each treatment were first normalized to total protein levels as determined by GAPDH and then to the appropriate control group.

Mitogen-activated protein kinase kinase (MEK) is upstream of ERK. At 30 min after treatment with the MEK inhibitor PD98059 (10 μM; Cell Signaling Technology, Inc., Danvers, MA, USA), the level of phospho-ERK (p-ERK) was detected by WB.
Samples of 10 μg total protein were separated in SDS-PAGE gel. The proteins were then transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked for 2 h at room temperature and then incubated with primary antibodies [rabbit anti-phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204; 1:1,000; Cell Signaling Technology, Inc.), rabbit anti-p44/42 MAPK (ERK1/2; 1:1,000; Cell Signaling Technology, Inc.) or rabbit anti-GAPDH antibody (1:5,000; Abcam)] overnight with gentle agitation at 4°C. The following day, the membrane was incubated with secondary antibodies [horseradish peroxidase-conjugated goat anti-rabbit IgG, (1:5,000; CoWin Biotech Co., Ltd., Beijing, China)] for 3 h at room temperature. Protein detection was performed using enhanced chemiluminescence.

The cells were washed with PBS and subsequently lysed using cell lysis buffer (Promega) with complete protease inhibitor mix (Roche). The cell lysate and control were run in SDS-PAGE gels (15%) respectively and transferred onto nitrocellulose membranes (Hybond-C Extra). Membranes were blocked with 5% milk in Tris-buffered saline plus Tween 20 (TBST) and exposed to antibodies against rabbit anti-apelin antibody (1:200, Abcam) and rabbit anti-GAPDH antibody (1:1000, Abcam). Blots were probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) secondary antibodies and visualized using a Bio-Rad Immun-Star Western C kit for signal detection.

The entire procedure is reported in the supporting information. Briefly, Western blotting was conducted to verify a subset of the key changes observed in protein expression of PPARG in SAT. The SAT was homogenized and hundred micrograms of total protein was subjected to 12% SDS/PAGE and transferred to a PVDF membrane (Millipore). The membrane was probed first with rabbit anti- PPARG antibody (Abcam, Cambridge, MA) and rabbit anti-GAPDH antibody (Abcam). The secondary antibody was goat polyclonal secondary antibody to rabbit IgG—H&L (HRP) (Abcam). Immunodetection using the Clarity Western ECL Substrate (Bio-rad, Hercules, CA) was performed according to the manufacturer’s instruction. The intensities of the bands were quantified [12 (link), 13 ] using ImageJ software [14 ] and the expression of PPARG protein was normalized to the expression level of GAPDH protein.

Total cell protein was extracted on ice using RIPA lysis buffer in the presence of freshly added protease and phosphatase inhibitors (Thermo), then quantified by the BCA method (Pierce). A total of 30 μg protein extract per lane was loaded onto a 14% SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Pierce). Nonspecific binding was blocked with 5% nonfat milk in PBS. The membrane was incubated with rat anti-IL-33 (R&D) or rabbit anti-phospho-STAT6 (Cell signaling) antibody overnight at 4°C. IRDye 800CW goat anti-rabbit IgG or goat anti-rat IgG (LI-COR) was used as secondary antibody, and rabbit anti-GAPDH antibody (Abcam) was used as an internal standard.