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.
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.