Rabbit anti β actin polyclonal antibody

The PC-3 and LNCaP cell lines were obtained from the Kunming Institute of Zoology, Chinese Academy of Sciences. Recombinant human IL-6 was purchased from R&D Systems (Minneapolis, MN). Gibco RPMI 1640 medium and fetal calf serum were purchased from Life Technologies (Grand Island, NY); HyClone 0.25% trypsinase from GE Healthcare Life Sciences (Piscataway, NJ); and DMSO from Sigma-Aldrich (St. Louis, MO). Anti–miR-21 inhibitor and anti–miR-21 negative control were purchased from ABI Biotech (USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA). The RNAiso Plus kit for RNA isolation, PrimeScript RT reagent kit for cDNA reverse transcription, and Perfect Real Time kit for quantitative real-time PCR (qRT-PCR) were purchased from Takara Bio (Mountain View, CA) along with the DL1000 DNA marker. Primers for PDCD4 and GAPDH were obtained from Sangon (China). Human anti-PDCD4 monoclonal antibody and rabbit polyclonal anti–β-actin antibody were purchased from Abcam (Cambridge, MA). Anti-rabbit IgG, HRP-linked antibody was purchased from Cell Signaling Technology (Danvers, MA). The BCA protein assay kit and the RIPA lysate buffer were purchased from Beyotime Institute of Biotechnology (China). In situ hybridization kits for HSP and DAB were purchased from LBP Biotech Company (China).

Tissue samples from the pericontusional area were washed with cold PBS and lysed in ice-cold RIPA buffer (1 ml per 100 mg tissue sample) containing protease inhibitors (Beyotime, China), followed by incubation on ice for 5 min and centrifugation at 4 °C for 15 min. The supernatant was collected, and protein concentration was measured with BCA (Beyotime, China). Equal amounts of protein from each sample were separated by electrophoresis on a 10% SDS-polyacrylamide gel (Beyotime, China), electrotransferred to a PVDF membrane (Millipore, USA), and blocked with 5% skimmed milk powder. Rabbit anti-β-actin polyclonal antibody (1:1000, ABCAm Biotechnology, UK), rabbit anti-HIF-1α polyclonal antibody (1:200, ABCAm Biotechnology, UK), rabbit anti-GLUT-1 polyclonal antibody (1:200, ABCAm Biotechnology, UK), and rabbit anti-GLUT-3 polyclonal antibody (1:200, ABCAm Biotechnology, UK) were used. Immunoblots were visualized by chemiluminescence using an ECL detection system (Thermo Fisher, MA, USA). The intensity of the bands was determined using the Quantity One 4.6.2 software.

Cells transduced with the Ad vector and the AAV vectors were lysed in Cell Lysis Buffer M (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). Lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (4‒12% polyacrylamide gel) and electrophoretically transferred to a membrane (Immobilon; Millipore). Blots were blocked and probed with a high affinity anti-HA rat monoclonal antibody (Roche, Mannheim, Germany, #11,867,423,001; 1:1,000) overnight at 4 °C or with an anti-β-actin rabbit polyclonal antibody (Abcam, Cambridge, UK, #ab8227; 1:5,000) for 2 h at 15–25 °C. Blots were then incubated for 2 h at 15–25 °C either with a peroxidase-conjugated goat anti-rat IgG F(ab')₂ fragment antibody (Jackson Lab, #112–036-062; 1:10,000) or a donkey anti-rabbit IgG F(ab')₂ fragment antibody (Abcam, #ab98440; 1:3,000) to detect bound anti-HA or anti-actin antibodies, respectively. Bound antibodies were visualized using a Chemilumi-One Chemiluminescent Kit (Nacalai Tesque, Kyoto, Japan).

Lysates were prepared from 16 P3 and P30 CBA/J mice cochleae sensory epithelia, as described above. Protein concentrations were determined by DC Protein assay (Bio-Rad) and equal amounts of proteins (3 μg/lane) from P3 and P30 tissues were resolved on Criterion 4–15% Tris-HCl SDS-PAGE gels (Bio-Rad) and transferred onto a nitrocellulose membrane (Amersham Biosciences). Blots were blocked at RT for 1 h in Tris-buffered saline/Tween 20 [50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 0.05% Tween 20] with 4.5% milk and then probed with respective primary antibodies including, anti-Dbn1 at 1:500, anti-Parvin at 1:800, anti-SPARC at 1:1000, and anti-Tmed10 at 1:1000 (all from Proteintech Group) with rocking O/N at 4 °C. Beta-actin was used as a protein loading control using anti-β actin rabbit polyclonal antibody (Abcam) for detection. Before adding secondary antibody, blots were washed 1X with TBS and 2X with 0.05% Tween/TBS. Membranes were then incubated with a donkey anti-rabbit horseradish peroxidase-conjugated secondary antibody at 1:5000 with rocking at RT for 1 h. Secondary antibody was removed and blots washed 1X with TBS, 2X with 0.05% Tween/TBS, and a final wash with TBS. Immunoreactive bands were developed using ECL (Amersham Biosciences) and Magic Mark XP (Invitrogen) was used as the protein standard to estimate relative mobilities.

Protein extractions and Western blotting were performed using a procedure reported previously³⁵ . The primary antibodies used were as follows: Anti-CD63 (8A12) mouse monoclonal antibody (Cosmo Bio, Tokyo, Japan), anti-β actin rabbit polyclonal antibody (#ab8227; Abcam, Cambridge, UK), anti-GAPDH rabbit polyclonal antibody (#ab9485; Abcam), anti-RAB5 (C8B1) rabbit monoclonal antibody (#3547, Cell Signaling Technology [CST], Danvers, MA, USA], anti-phospho-Akt (Ser473; D9E) rabbit monoclonal antibody (#4060; CST), anti-Akt (C67E7) rabbit monoclonal antibody (#4691; CST), anti-phospho-ERK1/2 (Thr202/Tyr204) rabbit monoclonal antibody (#4370; CST) and anti-ERK1/2 (137F5) rabbit monoclonal antibody (#4695; CST). The secondary antibodies used were an anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked antibody (#7074; CST) and anti-mouse IgG HRP-linked antibody (#7076; CST). The bound antibodies were visualised with the ImmunoStar Zeta or LD Chemiluminescence System (Wako). To release antibodies binding on membranes, Stripping solution (Wako) was used according to the manufacturer’s instructions.