Ab58641

Cells were grown in 10- or 15-cm dishes. At 80 to 90% confluency, plates were washed with cold PBS and collected in lysis buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% lauryl maltose neopentyl glycol, 1× protease inhibitor cocktail, and 1× phosphatase inhibitor]. The cell lysis solution was vortexed every 5 min for 30 min while kept on ice. The samples were spun down at 4°C for 15 min at 16,100g. The supernatant was then collected, and protein concentration was determined by Bradford assay. Total protein (500 to 1000 μg) was used for IPs with IgG control and specific antibodies [i.e., anti-IDH3α antibody (5 μg; ab58641, Abcam) and anti-cSHMT antibody (5 μg; ab186130, Abcam)]. Total protein (5 to 20 μg) was used as input. Samples were incubated at 4°C overnight while rotating. Dynabeads (50 μl per sample) (10004D, Life Technologies) were added, and after 30-min incubation at RT, beads were washed with PBS + 1× protease inhibitor and resuspended in 25 μl of NuPAGE LDS sample buffer (Life Technologies) + 5% β-mercaptoethanol. The samples were then heated at 95°C for 10 min and analyzed via WB.

The cells were treated with carnosine for 48 hours and then were lysed in Western
and IP lysis buffer containing PMSF for 5 minutes on ice, followed by
centrifugation at 13 000 × g for 25 minutes at 4°C. The
supernatant was harvested, and the protein concentration was quantified using a
BCA protein assay kit. Western blot analysis was carried out by standard
protocol. The following antibodies were used: rabbit anti-c-Myc antibody
(1:5000, ab32072), rabbit anti-PCNA antibody (1:1000, ab92552), rabbit
anti-Bcl-2 antibody (1:1000, ab32124), rabbit anti-SDHA antibody (1:1000,
ab137040), rabbit anti-IDH3A antibody (1:1000, ab58641), rabbit anti-MDH1
antibody (1:1000, ab180152), rabbit anti-ClpP antibody (1:1000, ab124822),
rabbit anti-ClpX antibody (1:1000, ab168338), rabbit anti-COX IV antibody
(1:1000, ab66739) (from Abcam Inc). Mouse anti-β-actin antibody (1:1000, AA128),
HRP-labeled goat anti-rabbit IgG (1:500, A0208), and HRP-labeled goat anti-mouse
IgG (1:500, A0216) were from Beyotime Institute of Biotechnology (Nanjing,
China).