Rabbit anti gapdh 14c10

Cell lysates were prepared and resolved as described^{12 (link)}. Primary antibodies used were: rabbit anti-PPP1R15A (10449-1-AP, 1:1000; Proteintech, Manchester, UK), rabbit anti-phospho-eIF2α (3597, 1:1000; Cell Signaling), mouse anti-actin (ab3280, 1:1000; Abcam), rabbit anti-ATF4 (C-20, 1:500; SantaCruz, Santa Cruz, CA, USA), mouse anti-total eIF2α (AHO0802, 1:1000; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA); guinea pig anti-insulin (ab7842, 1:1000; Abcam), rabbit anti-GAPDH (14C10) (2118, 1:1000; Cell Signaling), rabbit anti-UCP1 (ab10983, 1:1000; Abcam), rabbit anti-CHOP (1:1000; kind gift from Professor David Ron, Cambridge, UK), rabbit anti-alpha/beta tubulin (2148, 1:1000; Cell Signalling, Hitchin, UK).

Total proteins were extracted from wild-type and Human BAC2 Tg mouse brains and separated by 10% polyacrylamide gel electrophoresis. Proteins were transferred to PVDF membrane (Millipore IPVH00010) with AE-6687 HorizeBLOT system (ATTO). Primary antibodies and dilutions used were rabbit anti-Moesin (Msn, 1:1000, Cell Signaling 3146) and rabbit anti-GAPDH (14C10, 1:2000, Cell Signaling 2118). Secondary antibody was anti-rabbit IgG, horseradish peroxidase-linked (GE Healthcare NA9340V) diluted 1:5000. Signal bands were detected by using ECL Prime Western Blotting Detection Regent (GE Healthcare RPN2232) and images were captured on LAS-4000 Luminescent Image Analyzer (Fujifilm). Total density of each Msn and Gapdh protein band was determined with ImageJ (version: 2.0.0) software. For each sample, the ratio of Msn to Gapdh total density was calculated. Msn-to-Gapdh ratios were analyzed by Student’s t test for comparison of wild-type to Human BAC2 Tg brain samples (n = 3 for each brain regions).

Total protein extraction and electrophoresis were done according to the previous protocol [27 (link)]. The following primary antibodies were used: rabbit anti-PDCD10 (Atlas; 1:400), rabbit anti-GAPDH (14C10) (Cell Signaling; 1:1000), rabbit anti-phospho-Akt (p-Akt, Cell Signaling; 1:600), rabbit anti-RhoA (Santa Cruz Technology, 1:200), rabbit anti-phospho-myosin light chain 2 (Thr18/Ser19) (p-MLC2) (Cell Signaling; 1:1000) and mouse anti-GFAP (Sigma Aldrich; 1:5000), For semi-quantification of the blot, integrated optical density (IOD) of the bands was measured by Image J software. The relative expression of a target protein was calculated by the IOD ratio of the target protein to the housekeeping protein GAPDH, and the data were presented as percentage of the control.

Western blotting was used to verify the presence of HIF-1α in some samples. Cells were lysed and the protein content of the lysate quantified as previously described (Heikal et al. 2015 (link)). Thirty μg of cellular proteins were separated on a 10% SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Amersham/ GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). After blocking with 5% skimmed milk (for HIF-1α detection) or 5% bovine serum albumin; BSA (for GAPDH detection) for 1 h, membranes were incubated with the appropriate primary antibody overnight, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. HIF-1α and GAPDH (loading control) were detected using rabbit anti-HIF-1α (NB100-479, Novus biologicals, UK) and rabbit anti-GAPDH (14C10, Cell Signaling Technology, UK) at 1:500 and 1:1000 respectively and an anti-rabbit secondary antibody (A0545, Sigma Aldrich, UK) at 1:20,000 dilution. Protein bands were visualized by exposing membranes developed with the ECL reagent (Amersham/ GE Healthcare Life Sciences) to chemiluminescence film (Hyperfilm ECL, Amersham/ GE Healthcare Life Sciences). Bands were quantified using Image J software.

Samples containing equal amounts of protein (depending on the antibody, 5–50 µg) from lysates of cultured Osteosarcoma cells underwent electrophoresis on SDS-polyacrylamide gels and were transferred to polyvinylidenedifluoride (PVDF) membranes. The membranes were blocked in 3% BSA-PBS-0.05% Tween at room temperature for 1 h and blots were probed overnight at 4° C with the following primary antibodies : rabbit anti-MET (C-12), 1:500; Santa Cruz Biotechnology, rabbit anti-Akt #9272S, 1:1000; rabbit anti-P-Akt (S473) #9272S, 1:1000; rabbit anti-p44/42 #9102S, 1:1000; rabbit anti-P-44/42 MAPK (T202/Y204) #4370S, 1:2000; or rabbit anti-GAPDH 14c10, 1:2000; Cell Signaling Technologies, Beverly, CA, to detect proteins of interests. After incubation, the membranes were washed 3 times with washing buffer (PBS containing 0.05% Tween) for 5 min. Membranes were then incubated for 1 h with 1:10,000 diluted secondary antibody (goat-anti-rabbit sc-2004 #J1512, 1:10000; Santa Cruz Biotechnologies, Santa Cruz, CA) at room temperature. Specific proteins were detected using SuperSignal^® WestDura Extended Duration Substrate (ThermoScientific, Rockford, USA) and a G-Box (Syngene, Cambridge, UK) after washing. Pictures were analysed thanks to the ImageJ software. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal loading control.