Anti mct4 antibody

For studying the kinetics of mitochondrial ROS production, FACS-sorted CD44⁺CD117⁺ and CD44⁺CD117⁻ cells were maintained at 37°C in DMEM medium (Sigma Aldrich) supplemented with 10% FBS, 1% sodium pyruvate, and 1% Hepes (Lonza). Cultures were pretreated with 2 mM pargyline, and then labeled with 20 nM H₂-MTR. ROS production was measured using an LSM510 confocal laser microscope (Zeiss, Jena, Germany) equipped with a 37°C, 5% CO₂ incubator using Helium Neon (543 nm) and Argon (488 nm) lasers. Laser intensity, pinhole aperture, and photomultiplier parameters were standardized to allow comparison of signals obtained in different samples; images were recorded at 1 min intervals for 60 min. The mean fluorescent signal of H₂-MTR in mitochondria of individual cells was quantitated with the Zeiss Histogram software tool, and expressed as ΔF/F₀ as above. To evaluate MCT4 expression, FACS-sorted CD44⁺CD117⁺ and CD44⁺CD117⁻ cells were stained with anti-MCT4 antibody (1:100; Santa Cruz Biotechnology, Dallas, TX); nuclei were stained with TOPRO3 (1:10,000; Invitrogen), and images recorded by confocal microscopy.

Puromycin dihydrochloride (cat. No. #sc-108071), control shRNA plasmid-A (#sc108060), MCT4 shRNA plasmid (h2) (#sc-45892-SH) and anti-MCT4 antibody (anti-rabbit, monoclonal, #sc-50329) were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-phospho-p44/42 MAPK antibody (p-ERK; anti-rabbit, monoclonal, #4370), anti-p44/42 MAPK antibody (ERK; anti-rabbit, monoclonal, #4695), horseradish peroxidase (HRP)-conjugated IgG antibody (goat anti-rabbit, monoclonal, #7074), HRP-conjugated IgG antibody (goat anti-mouse, monoclonal, #7076), Alexa Fluor 488-conjugated IgG (H+L) F(ab′)2 fragment (goat anti-rabbit, monoclonal, #4412), and Alexa Fluor 488-conjugated IgG (H+L) F(ab′)2 fragment (goat anti-mouse, monoclonal, #4408) were purchased from Cell Signaling Technology (Danvers, MA). Anti-VR1 antibody (TRPV1; anti-mouse, monoclonal, #ab203103), anti-CGRP antibody (anti-goat, polyclonal, #ab36001) and Alexa Fluor 647-conjugated IgG H&L (donkey anti-goat, monoclonal, #ab150135) were purchased from Abcam (Cambridge, MA, USA).