Mouse anti gst

For GST pull-down assays, HEK 293T cells were co-transfected with plasmids expressing GST or GST fusion proteins together with GFP or GFP-DLC1 constructs. Forty-eight hours after transfection, cells were lysed with 1X RIPA buffer (EMD Millipore) (0.05M Tris-HCl pH 7.4, 0.15mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1mM EDTA) containing protease and phosphatase inhibitors. Supernatants were collected after centrifugation at 14000 rpm for 10 min at 4° C, and protein was quantified with a BCA kit (Thermo Scientific) following manufacturer’s instructions. One and one-half milligrams of protein from each cell extract were used for pull-down assays by adding 30 μl of glutathione Shepharose-4B slurry (GE Healthcare) and rotating 4 hours at 4° C. Pellets were extensively washed for 3–4 times with 1X RIPA buffer and incubated with 40 μl loading buffer. After separating protein samples by SDS-PAGE, immunoblotting was used to detect protein signals with mouse anti-GST (Santa Cruz Biotechnology) or mouse anti-GFP (Covance) antibodies. Horseradish peroxidase-conjugated anti-mouse IgG (GE Healthcare) was used as the secondary antibody. Immunocomplexes were visualized with an ECL kit (Amersham).

Whole cell lysates were prepared from frozen cell pellets by lysis
in trichloroacetic acid as described previously [20 (link)]. Protein concentrations were measured by a
bicinchoninic acid assay (Thermo Scientific # 23225), and equal amounts (10
or 20 μg) were loaded per lane. Proteins were resolved by
SDS–PAGE and transferred to PVDF membranes (Immobilon-P; Millipore)
in a submerged tank. Membranes were blocked (1 hr, room temperature) in TTBS
(0.2% Tween-20, 20 mM TRIS-HCl, 500 mM NaCl, pH 7.5) containing 5% non-fat
milk, and then probed with antibodies in the equivalent solution. Primary
antibodies were mouse anti-HA (1:1000, Covance #MMS101R), mouse anti-V5
(1:5000, Invitrogen #46–0705) mouse anti-phospho-p44/42 (1:1000, Cell
Signaling Technology #9101), mouse anti-GST (1:1000, Santa Cruz
Biotechnologies #sc-138), rabbit anti-myc (1:200, Santa Cruz Biotechnologies
#sc-789), and rabbit anti-G6PDH (1:100000, Sigma #A9521). HRP-conjugated
secondary antibodies were goat anti-rabbit (1:3000, Jackson ImmunoResearch
#111–035-144), and goat anti-mouse (1:3000, BioRad #170–6516).
Enhanced chemilluminescent detection used a BioRad Clarity substrate
(#170–5060). Densitometry was performed using ImageJ software.