Ab56676

The anti-p65 antibody (sc-372), the anti-PDGFR-β antibody (sc-432), the anti-β-actin antibody (sc-517582) and the anti-NG2 antibody (sc-166251) were obtained from Santa Cruz Inc. The antibodies anti-CD54 (ICAM-1) (555511), anti-CD62E (E-selectin) (551145), anti-CD106 (VCAM-1) (555647) and IgG1-κ Isotype Control (555749) were purchased from BD Biosciences (Heidelberg, Germany). Peroxidase-labeled anti-rabbit antibody (NIF 824) and peroxidase-labeled anti-mouse antibody (NIF 825) were obtained from GE Healthcare (Freiburg, Germany). The anti-HO-1 antibody (ADI-SPA-895) was from Enzo Life Sciences (Lörrach, Germany). The anti-α-tubulin antibody (ab56676), the anti-myeloperoxidase (MPO) antibody (ab9535), the anti-ICAM-1 antibody (ab124760), the anti-8-OHDG antibody (ab10802), the anti-eNOS antibody (ab5589), the anti-VCAM-1 antibody (ab134047), the anti-CD68 antibody (ab1252212) and the secondary biotinylated goat anti-rabbit antibody (ab64256) were purchased from Abcam.

Proteins were extracted from spinal cord segments collected at 24 h and 7 days post-injury, resolved in Novex 4-12 % BisTris gels (NuPage, Life Technologies, Carlsbad, CA, USA), and transferred onto a PVDF membrane (Roche, Basel, Switzerland) according to standard protocols. Blots were then probed with primary antibodies targeting Ly6G/Gr-1 (1/500, MCA2387, AbD Serotec, Kidlington, United Kingdom) and CD11b (1/500, ab75476, Abcam, Cambridge, United Kingdom) at 4 °C overnight. Secondary antibodies coupled to HRP (1/3000, ab97057, Abcam, Cambridge, United Kingdom) were applied for 1 h, and blots were revealed with a chemiluminescent substrate (ThermoScientific, Waltham, MA, USA) and imaged with the ImageQuant 350 scanning system (cooled-CCD camera, GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). α-tubulin detection (1/10.000, ab56676, Abcam, Cambridge, United Kingdom) was used as internal standard. Expression levels were quantified with ImageMaster 1D Prime Software (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).

A goat anti-cathepsin D antiserum was purchased from Santa Cruz Biotechnology (sc-6487 Dallas, USA). Rabbit anti-prosaposin antiserum was kindly provided by Dr Morales (Mc Gill University, Montreal, Canada) (Lefrancois et al., 2003 (link)). The rabbit anti-LAMP-1 (ab-24170) antiserum and anti-tubulin monoclonal antibody (ab-56676) were obtained from Abcam (USA), anti-Glial-Fibrillary Acidic Protein (GFAP) monoclonal antibody produced in mouse was obtained from Sigma-Aldrich (G-3893, USA). HRP-conjugated anti-goat IgG antiserum was obtained from H&L (401515), the HRP-conjugated anti-rabbit IgG fraction of antiserum was obtained from Sigma-Aldrich (A 9169) and anti-mouse IgG (whole molecule)-peroxidase was purchased from Sigma-Aldrich (A 9044). Chemiluminescent reagents were obtained from Pearce (Rockford, USA).

Cells were lysed using a lysis buffer (Beyotime, Shanghai, China) and then protein concentration was quantified using a BCA protein assay kit (Beyotime). Then the protein samples were subjected to a conventional western blot analysis. After separation on 10% SDS PAGE gel, the proteins were subsequently transferred onto PVDF membrane and blocked with 5% nonfat milk for 1 h. Membranes were probed overnight at 4°C with primary antibodies: anti‐LC3B (1:3000, ab51520, Abcam, Cambridge, MA, USA), anti‐p62/SQSTM1 (1:1000, #8025, Cell Signaling), anti‐DICER (1:200, H‐212; Santa Cruz Biotechnology), anti‐active caspase‐3 (1:1000, ab2302, Abcam), anti‐tubulin (1:1000, ab56676, Abcam), and anti‐β‐actin (ab189073, 1:1000, Abcam). Band signals were visualized using HRP‐linked secondary antibodies (Abcam) and the ECL Western blotting substrate (Promega, Madison, WI, USA).

Western blot analyses were carried out as described (24 (link)) using antibodies against U2AF35 (10334–1-AP, Protein Tech Group), U2AF65 (U4758, Sigma), actin (ab37063, Abcam), tubulin (ab56676, Abcam) and CAPERα (PA5–31103, Thermo Fisher Scientific). AntiXpress antibodies were purchased from LifeTechnologies (R910–25). Antibodies against PUF60, hnRNP C and hnRNP I (PTB) were a generous gift of Adrian Krainer, Gideon Dreyfuss and Christopher Smith, respectively.

Liver homogenates and mitochondrial fractions were obtained in RIPA buffer (Sigma-Aldrich, SLM, USA) and subjected to denaturing SDS-PAGE under reducing conditions. Total protein concentrations were determined by the Bradford method and equal amounts (30 μg) were separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and blocked for 1 h in TBST buffer (20 mM TRIS, pH 7.5; 500 mM NaCl; 0.5% Tween 20) containing 5% nonfat milk. Membranes were then washed and incubated in the presence of mouse anti-GABA-T antibody (Rb mAb to ABAT, ab108249, Abcam, Cambridge, UK) diluted 1/10,000 in TBST overnight at 4°C. As controls, in the case of homogenates, membranes were incubated in the presence of mouse anti-tubulin antibody (ab56676, Abcam, Cambridge, UK) diluted 1/1,000 or in the case of mitochondrial fractions in the presence of rabbit anti-VDAC1/Porin antibody (ab15895, Abcam, Cambridge, UK) diluted 1/1,000. After washing, membranes were incubated with secondary antibodies conjugated to alkaline phosphatase (1/5,000). Bands were revealed using AP conjugate substrate kit (Bio-Rad, CA, USA). Densitometric analysis was performed using the Image Lab Software (v 3.0, Bio-Rad, CA, USA).