Rabbit anti ldha

The equations should be inserted in editable format from the equation editor. Five human colon cancer cell lines, HT-29, SW620, HCT116, SW480, and DLD-1, and one normal human colon epithelial cell line, CRL-1790, were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/mL penicillin G, and 100 μg/mL streptomycin (Thermo Fisher Scientific, Inc., Rockford, IL, USA) at 37°C with a humidified air containing 5% CO₂. The Cisplatin-resistant colon cancer cell line SW480 was established according to previous description (30 (link)). Cells were maintained in Dulbecco modified Eagle medium in a 5% CO₂ incubator at 37°C. The acquired Cisplatin-resistant cells were reselected by treating with Cisplatin at 30 μM every 2 months. The rabbit anti–hexokinase 2 (HK2) (#2867); rabbit anti-LDHA (#3582), and rabbit anti–β-actin (#4970) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Cisplatin and oxamate were purchased from Sigma–Aldrich (Shanghai, China).

Briefly, SiHa cells were lysed in cell lysis buffer with intermittent vortexing for 1 hour. This was followed by centrifugation of the lysate at 15000 rpm for 10 min at 4 °C and supernatant was collected. The protein samples were quantified using Bradford assay and the volume corresponding to 70 µg of protein was mixed with 4X loading dye and boiled at 95 °C for 7 minutes, then loaded onto a 12% PAGE. Proteins in the gel were transferred onto a PVDF membrane (GE), blocked with 5% milk/PBS-T, and then probed with primary antibodies at appropriate dilutions. Primary antibodies used included: mouse anti-β-actin (1:400, Santacruz, Cat #Sc 47778), mouse anti-E7 (1:400, Santacruz, Cat#Sc- 6981), mouse anti-E6 (1:400, Santacruz, Cat#Sc 460), mouse anti- pRB (1:500, Cell Signalling, Cat# 9309), rabbit anti- p14ARF (1:400 Santacruz, Cat# Sc 53639), rabbit anti-PARP (1:1000, Cell Signalling, Cat# 9542), rabbit anti-RIP1 (1:1000, Cell Signalling, Cat# 4926), rabbit anti-LDHA (1:1000, Cell Signalling, Cat# 2012), Cyclophilin A (1:3000, Abcam, Cat# ab58144). Secondary antibodies included: Goat anti-rabbit HRP (Sigma Aldrich Cat# A9169) and Goat anti-mouse HRP conjugates (Sigma Aldrich, Cat# A4416). Proteins were visualized using chemiluminescence (Immobilon, Millipore). All Western blots were done in triplicates.

Mouse anti-c-myc monoclonal antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse anti-β-actin was purchased from Millipore Corporation (Billerica, MA, USA). Rabbit anti-HIF-1α, rabbit anti-glut1, rabbit anti-LDHA, rabbit anti-pyruvate kinase isozyme M2 (PKM2), rabbit anti-2-oxoglutarate dehyrogenase E1 component (OGDH), and rabbit anti-glutaminase (Gls) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against ARV σA and p17 proteins were from our laboratory stocks [8 (link),16 (link)]. The secondary antibodies, including goat anti-mouse IgG (H + L) and goat anti-rabbit IgG (H + L) HRP conjugate and rhodamine-labeled affinity purified antibody to mouse IgG (H + L) were purchased from Kirkegaard and Perry Laboratories (Washington, DC, USA).

Cells were fixed in cold 4% paraformaldehyde in PBS for 10 min. NPCs and neurons were permeabilized at room temperature for 15 min in 0.2% TritonX-100 in PBS. Samples were blocked in 5% BSA with 0.1% Tween 20 for 30 min at room temperature. The following primary antibodies and dilutions were used: goat anti-Sox2 (Santa Cruz Biotechnology, Dallas, TX), 1:200; mouse anti-Nestin (EMD Millipore, Temecula, California), 1:200; rabbit anti-βIII-tubulin (Covance, San Diego, CA), 1:200; mouse anti-βIII-tubulin (Covance), 1:200; rabbit anti-GFAP (Dako, Carpinteria, CA) 1:200; mouse anti-MAP2AB (Sigma-Aldrich, St. Louis, MO), 1:200; rabbit anti-LDHA (Cell signaling, Danvers, MA), 1:200, and rabbit anti-HK2 (Cell signaling), 1:200. Secondary antibodies were Alexa donkey 488 and 568 anti-mouse, rabbit and goat (Invitrogen, Carlsbad, CA), used at 1:1000. Nuclear staining was done with Hoechst (Invitrogen).