Ab90582

The cells were collected and lysed in the lysis buffer which was mixed with 0.1% sodium dodecyl sulfate (SDS), 50 mM TrisHCl, sodium deoxycholate, 150 mM NaCl, 1% NP-40, and a protease inhibitor (PH = 7.5). Then, the protein samples were loaded to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (PVDF). After blocking with 5% non-fat dry milk for 1 h, the membranes were incubated with the specific primary antibody overnight. After washing in TBST for three times in 15 min, the membranes were then incubated with the secondary antibodies (peroxidase-labeled anti-mouse and anti-rabbit antibodies) for 1 h. Briefly, the primary antibodies were utilized at a dilution of 1:2,000, while secondary antibodies were diluted with 1:5,000. After washing in TBST for three times in 15 min, the membranes were finally visualized in an ECL + plus^TM Western Blotting system kit (cat. no. RPN2232; Amersham). The detailed information of the primary antibodies was summarized as follows: KAT2A (abcam, ab217876); MCT1 (abcam, ab90582); β-Actin (CST#4970).

Immunohistochemical analysis was performed as previously described [79 (link)]. Briefly, deparaffinized and pretreated slides were incubated for 30 min at 37 °C with MCT4 and HCAR1 (1:1000; AB90582, Abcam, Cambridge, UK) antibody. Immunostaining specificity was assayed omitting antibodies.

Briefly, for Western blot analysis, 30 μg of protein was loaded onto a 12% polyacrylamide gel, MiniPROTEAN^® TGXTM (Bio-Rad, Milan, Italy), followed by electrotransfer to a nitrocellulose membrane, TransBlot^® TurboTM, using TransBlot^® SE Semi-Dry Transfer Cell (both from Bio-Rad, Milan, Italy) (18 (link)). Subsequently, the membrane was blocked in Odyssey Blocking Buffer (Licor, Milan, Italy) for 1 h at room temperature. After blocking, the membrane was washed three times in phosphate-buffered saline (PBS) for 5 min and incubated with primary antibodies against MCT1 (1:1,000; AB90582), MCT4 (1:1,000; AB74109), β-catenin (1:500; AB16051), E-cadherin (1:500; AB76055, all from Abcam, Cambridge, UK), and β-actin (1:1,000; anti-mouse, cat. no. 4967S; Cell Signalling Technology, Milan, Italy) overnight at 4°C. The next day, the membranes were washed three times in PBS for 5 min and incubated with infrared anti-mouse IRDye800CW (1:5,000) and anti-rabbit IRDye700CW secondary antibodies (1:5,000) in PBS/0.5% Tween-20 for 1 h at room temperature. All antibodies were diluted in Odyssey Blocking Buffer. The blots were visualized using Odyssey Infrared Imaging Scanner (Licor, Milan, Italy), and the protein levels were quantified by densitometric analysis. Data were normalized to the expression of β-actin.

Proteins of xenografts and cells were extracted using Pierce™ IP lysis buffer (87787; Thermal Fisher). 40 μg of the protein was resolved in each well of 10% SDS-PAGE and transferred to Polyvinylidene Fluoride (PVDF) membranes. Then, the blots were blocked by 5% fat free milk and incubated with anti-HIF-1α (1:1000; ab1; Abcam), anti-PGC1α (1:1000; ab106814; Abcam), anti-MCT1 (1:3000; ab90582; Abcam), anti-MCT4 (1:1000; ab234728; Abcam), anti-CD174 (1:2000; ab235831; Abcam), anti-HMGB1 (1:1000; ab18256; Abcam), anti-E-cadherin (1: 20000; ab40772; Abcam), anti-N-cadherin (1:10000; ab76011; Abcam), anti-Bcl-2 (1:1000; ab32124; Abcam), anti-p-GSK (1:10000; ab75814; Abcam), anti-GSK (1:8000; ab32391; Abcam), anti-ERK (1:10000; ab184699; Abcam), anti-p-ERK (1:1000; ab201015; Abcam), anti-JNK (1:1000; ab179461; Abcam), anti-p-JNK (1:5000; ab124956; Abcam), anti-p38 (1:5000; ab170099; Abcam), anti-p-p38 (1:1000; ab178867; Abcam) primary antibodies at 4 °C overnight. The day after, rabbit anti mouse IgG secondary antibody (1:2000; ab6728; Abcam) was incubated with the membranes at room temperature for 2 h. GAPDH was used as control. Finally, protein bands were visualized using the Ultra High Sensitivity ECL Substrate Kit (ab133409; Abcam).

Larval tissues were stained using standard immunohistochemical procedures. Briefly, discs were dissected in PBS, fixed at room temperature for 20 min in 3.7% formaldehyde/PBS and washed in 2% Triton-X100/PBS. All subsequent incubations were performed in 2% Triton X-100/PBS at 4 °C. Samples were mounted either in Vectashield or Vectashield containing DAPI (Vector Labs). The following primary antibodies were used: mouse anti-dNR2^{15 (link)} (1:100, from Ann-Shyn Chiang, mouse anti-dlg1 (1:50, 4F3, DSHB, University of Iowa, Iowa City, IA, USA), mouse anti-GFP (11814460001, Roche), mouse anti-MMP1 (1:50, 5H7B11, DSHB, University of Iowa, Iowa City, IA, USA), rabbit anti-cleaved DCP1 (Asp216) (1:200,Cell Signaling), mouse p-JNK (1:100, 9255,Cell Signaling Technology Inc., Danvers, MA, USA), PHA-555 (Phalloidin-555, A34055, Invitrogen/Molecular probes), mouse polyclonal anti-MCT1 (1:100, ab90582, Abcam), rabbit polyclonal anti-Myc (1:100, d1-717, sc-28207 Santa Cruz Biotechnology), rabbit polyclonal anti-p-PDHE1 (1:200, Pyruvate Dehydrogenase E1-alpha subunit (phospho S293), ab92696, Abcam). Note, the phosphorylation site surrounding S296 of human PDHE1 is conserved in Drosophila PDHE1, which is encoded by lethal(1)G0334 (CG7010) (e-value 5e-36, query coverage of 99%). Fluorescent secondary antibodies (1:2000, FITC-, Cy3- and Cy5-conjugated) were obtained from Jackson Immunoresearch.