Ab110025

RIPA buffer (Beyotime, Shanghai, China) was utilized to extract proteins from the treated DRG neurons. BCA Protein Assay Kit (Thermo, Cat no. 233225) was applied to examine the concentration of protein in each group. The extracted proteins (30 μg) were separated by10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were then blocked with 5% non-fat dry milk and incubated with primary antibodies at 4°C overnight. After washing, the membranes were incubated with the secondary antibody (1/3000, Abcam, ab205719). The blots were obtained by using the enhanced chemiluminescence (Thermo Scientific). The primary antibodies contain anti-GAPDH (1/2000, Santa Cruz), anti-NADPHase (1/1000, Abcam, ab127942), anti-HK2 (1/1000, Abcam, ab104836), anti-PFK1 (1/1000, Abcam, ab170868), anti-PK2, (1/1000, Abcam, ab76747), anti-Glut1 (1/1000, Abcam, ab40084), anti-LDHA, (1/1000, Abcam, ab125683), anti-PDK1 (1/1000, Abcam, ab110025), anti-Bax, (1/1000, Abcam, ab53154) and anti-Bcl-2 (1/1000, Abcam, ab196495), antibodies.

Paraffin sections were dewaxed, hydrated, immersed in 3% hydrogen peroxide solution for 15 minutes, and washed with PBS. Antigens were retrieved with 0.1 M sodium citrate. The sections were blocked with goat serum and incubated for 30 minutes at 37°C. After removal of serum, without washing, PI3K (ab151549, Abcam, USA), PDK1 (ab110025, Abcam, USA), AKT (ab8805, Abcam, USA), and MBP (ab40390, Abcam, USA) antibodies were added and incubated at 4°C overnight. The sections were washed with PBS and incubated with fluorescence-labeled secondary antibody for 30 minutes at 37°C, followed by PBS washes. DAPI-stained nuclei were added and incubated at room temperature for 10 minutes, followed by PBS washes. The sections were mounted with neutral resin and observed with a fluorescence microscope.

The paraffin embedded sections were cleared and the sections were incubated with 0.1% Pronase (Roche #165 921) in 0.1% CaCl2 pH 7.8. at 37C for 10 minutes. They were blocked with 3% H2O2 in TBS for 10 mins., washed then blocked with Dako Biotin Blocking System (Dako X0590). After washing, they were further blocked with 10% Normal Rabbit Serum for 10 mins at room temperature (RT) and incubated firstly with PDK1 (Abcam, ab110025) dilution 1:50, and GLUT-1 (Abcam, ab652) dilution 1: 1000 for 1h at RT, then with biotinylated Rabbit Anti-Mouse (Dako, E-0354) at 1/100 for 30 mins. at RT, and finally with Strep-ABC complex (Dako, K-0377) at 1/100 for 30 mins. at RT. The sections were developed with AEC substrate kit (vector lab, SK-4200) at RT for 20 mins., counterstained with haematoxylin and mounted with DAKO aqueous mount (Dako, 003181).

Antibodies anti-human proteins used for Western blot (WB), immunocytochemistry (ICC) and immunohistochemistry (IHC-P) were: mouse monoclonal anti-VEGFA (sc-7269 by Santa Cruz Biotechnology, Dallas, TX, USA); rabbit polyclonal anti-PHD3 (ab30782 by Abcam, Cambridge, UK); mouse monoclonal anti-PDK1 for WB and ICC (ab110025 by Abcam); rabbit polyclonal anti-PDK1 for IHC-P (sc-28783 by Santa Cruz); rabbit polyclonal anti-PFKFB4 (PA5-15475 by Thermo Scientific, Waltham, MA, USA); mouse monoclonal anti-HIF-1α for WB (610959 by BD Biosciences, Franklin Lakes, NJ, USA); rabbit polyclonal anti-HIF-1α for ICC and IHC-P (NB100-479 by Novus Biologicals, Littleton, CO, USA); rabbit polyclonal anti-N-Myc (23960002 by Novus Biologicals).

Cells were lysed in 1 × SDS sample buffer. Kidney tissue was homogenized by a polytron homogenizer (Brinkmann Instruments) in RIPA lysis buffer on ice. The supernatants were collected after centrifugation at 13,000 × g at 4 °C for 30 min. Protein concentration was determined by bicinchoninic acid protein assay. An equal amount of protein was loaded into 10% or 15% wt/vol. SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. The primary antibodies were as follows: anti-PPARα (ab24509, Abcam, Cambridge, MA, USA), anti-CPT1α (ab128568, Abcam), anti-ACADL (ab196655, Abcam), anti-HK2 (ab76959, Abcam), anti-LDH (3558, Cell Signaling Technology, Danvers, MA, USA), anti-PDK1 (ab110025, Abcam), anti-HIF-1α (ab1, Abcam), anti-PHD2 (4835, Cell Signaling Technology), and anti-tubulin (T6074, Sigma Aldrich). Western blot was performed at least three times independently. Chemiluminescence is applied for detecting proteins on western blot membranes. The enhanced chemiluminescent ECL substrate (32209, Thermofisher Scientific, Carlsbad, CA, USA) enables immunodetection of horseradish peroxidase (HRP)-conjugated secondary antibodies using an imaging system. Quantification was performed by measurement of the intensity of the signals with the aid of Image J software package.

The cells were harvested and lysed with RIPA buffer containing protease and phosphase inhibitors (Sigma-Aldrich). After centrifugation at 12,000 rpm for 10 min at 4 °C, the supernatants were collected and quantified using the Bradford assay (Bio-Rad). The proteins were then separated using SDS-PAGE and transfected to PVDF membranes (Bio-Rad). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies, including anti-PDK1 (ab110025; Abcam), anti-p-Akt (ab38449), anti-Akt (ab32505), anti-p-mTOR (ab109268), anti-Bcl2 (ab692), anti-caspase 3 (ab32351), anti-caspase 9 (ab32539), and anti-GAPDH (ab8245) followed by incubation with the secondary HRP-conjugated antibody (Cell Signaling). The bands were scanned using an enhanced chemiluminescence system and protein intensity was quantified with Image-Pro Plus 6.0 software (Media Cybernetics). GAPDH was used as the internal control.