Mouse anti α tubulin

Primary antibodies used in this study were as follows: mouse anti-USP4 (66822; Proteintech1:1000 dilution for WB, 1:300 dilution for IHC); mouse anti-BRCA1 (sc-6954; Santa Cruz; 1:300 dilution for WB); rabbit anti-BRCA1(ab16780; Abcam;1:400 dilution for IHC); rabbit anti-BRCA1 (ab191042; Abcam;1:1000 dilution for WB); rabbit anti-BARD1 (NB100; Novus Biologicals; 1:1000 dilution for WB); mouse anti-BARD1 (sc-74559; Santa Cruz; 1:500 dilution for WB); mouse anti-Flag (F3165; Sigma; 1:5000 dilution for WB); rabbit anti-HA (3924 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-Myc (M4439; Sigma;1:5000 dilution for WB); mouse anti-ubiquitin (3936 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-α-His (2366 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-α-Tubulin (3873 S; Cell Signaling Technology; 1:3000 dilution for WB), rabbit anti-β-actin (AC026; ABclonal; 1:1000 dilution for WB); rabbit anti-GAPDH(5174 S; Cell Signaling Technology; 1:5000 dilution for WB). For WB, western blots were detected and analyzed using ChemiDoc Imaging system (Bio-Rad). The original WB blots were included in the supplementary information.

To assess the cytotoxicity of MβCD, the viabilities of iSLK cells treated with MβCD were determined by counting Trypan blue-stained cells 5 days post-treatment using a light microscope. Cell viabilities were defined relative to control cells (untreated with MβCD). The half-maximal cytotoxic concentration (CC₅₀) was calculated from dose-response curves with GraphPad Prism software.
To assess the effect of MβCD on iSLK cell proliferation, 1 × 10⁵ cells were treated with MβCD for 5 days at four different concentrations (0, 0.5, 1, 5 mM) respectively. The cells were collected every day and assayed by MTT.
To assess the effect of MβCD on Lipid raft (LR) or microtubule organization, iSLK cells were treated with 1mM MβCD for 1–5 days and analyzed by IFA. LRs were stained by CTB-555 (Life Technologies, 1:100 dilution) before cell fixation. While, for microtubule staining, the cells were fixed and punched first, and then were stained by mouse anti-α-tubulin (Cell Signaling, 1:100 dilution) and Fluor Alexa-488-conjugated anti-mouse IgG (Life Technologies, 1:500 dilution). The nuclei were stained by Hoechst 33258 (0.1 μg/ml). Slides were examined with a Zeiss LSM780 confocal laser scanning system (63×oil).

The HUVECs were lysed with the RIPA solution (0.1% SDS, 150 mM NaCl, 1.0% Triton X-100, 10 mM Tris, 5 mM EDTA pH 8.0), to which we added a protease and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). In each sample, the protein concentration was evaluated using a Bradford assay. Proteins (25 µg) were separated by 4–15% precast polyacrylamide gel (Bio-Rad, Hercules, CA, USA), and then transferred to a 0.2 mm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). A blocking buffer (Bio-Rad, Hercules, CA, USA) was used to block the membrane, which was then incubated overnight with primary antibodies. Mouse anti-phospho-p38 (Cell Signaling), rabbit anti-phospho-NF-kB (Cell Signaling), mouse anti-ICAM-1 (Cell Signaling), mouse anti-β-actin, mouse anti-α-tubulin, and rabbit-anti-GAPDH (Cell Signaling) were used as the primary antibodies. Anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies were used as the secondary antibodies (The Jackson laboratory, Bar Harbor, ME, USA). A Uvitec Imager (UVItec, Cambridge, UK) was used to distinguish the protein bands, using a chemiluminescence substrate (Bio-Rad), that were then quantified using ImageJ software. Each measure was normalized versus β-actin, α-tubulin, or GAPDH.

For immunostaining, samples were fixed with 3.7% formaldehyde (Sigma-Aldrich) for 30 min, permeated with Triton X-100 (0.5%) for 30 min, and incubated overnight with primary monoclonal antibodies mouse anti-α-Tubulin (1:50, Cell Signaling Technology 3873, USA) and mouse anti-β-Tubulin (1:50, Cell Signaling Technology 86298) and then 2 h with secondary antibodies anti-mouse IgG (H+L) and F (ab')2 Fragment (Alexa Fluor^® 555 or 488 Conjugate, Thermo Fisher Scientific, USA) (1:50). Nuclei were labeled with DAPI (1:100) or PI (10 mg/mL) from Sigma-Aldrich. The slides were mounted with Vecta-Shield from Vector Laboratories (USA) and analyzed by laser scanning confocal microscopy from Leica TCS SP8 (Germany).

Antibodies used for immunoblotting, IP, and IHC staining were as follows: Rabbit anti ZHX2 antibody (Genetex, 112232), Rabbit anti HIF1α (Cell Signaling, 36169), Rabbit anti HIF1β (Cell Signaling, 5537), Rabbit anti VHL (Cell Signaling, 68547), rabbit anti HA tag (Cell Signaling, 3724), mouse anti α-Tubulin (Cell Signaling, 3873), mouse anti Ubiquitin (Santa Cruz, sc-8017), and Rabbit anti NF-κB p65 (Cell Signaling, 8242S). Peroxidase conjugated goat anti-mouse secondary antibody (31430) and peroxidase conjugated goat anti-rabbit secondary antibody (31460) were from Thermo Scientific.

Co-IP and western blotting were performed as previously described [19 (link)] with minor modifications (Supplementary Materials and Methods). In Fig. 6A, B, D–G, M, N, the intensity of each lane was measured by ImageJ software (https://imagej.nih.gov/ij/). Following antibodies were used for Co-IP: rabbit anti-Ddx20 antibody (10 µg, homemade, Immunogen is 722-740 aa of mouse Ddx20), mouse anti-FLAG M2 (1:1,000, mouse, Sigma, F1804), normal rabbit IgG (10 µg, MBL, Cat#PM035). Following antibodies were used for western blotting: rabbit anti-HA (1:1,000, MBL, Cat#561), mouse anti-FLAG M2 (1:1,000, mouse, Sigma, F1804), mouse anti-Myc (1:1000, DSHB, 9E10), rabbit anti-Ddx20 antibody (1:1,000, homemade), mouse anti-Olig2 antibody (1:1,000, Millipore, Cat#MABN50), mouse anti-SMN (1:1000, BD Biosciences, Cat#610646), mouse anti-Gemin2 (1:1000, BioLegend, Cat#862902), mouse anti-Gemin6 (1:1000, BioLegend, #862302), mouse anti-β-actin (1:2,000, Sigma-Aldrich, Cat#AC-15), mouse anti-α-Tubulin (1:2000, Cell Signaling Technology, Cat#3873), horseradish peroxidase (HRP)-labeled anti-rabbit IgG, HRP-labeled anti-mouse IgG (1:2,000, Cell Signaling Technology, Cat#7074, 7076).

The rats in each group (n = 3) were sacrificed at seven and 14 days after MCAO. Tissues in the ipsilateral hemisphere (excluding the infarct area) were selected for Western blotting. Under deep anesthesia, the brain tissue was rapidly removed and dissected, and protein was homogenized in a cell lysis buffer (Fermentas, Burlington, ON, Canada) containing a complete protease inhibitor cocktail (Thermo, Rockford, AL, USA). Next, 12% sodium dodecyl sulphate-polyacrylamide gels were used to separate equal amounts of protein from each sample which were electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore), The membranes were incubated with rabbit anti-BDNF (1:2000; Abcam, 15 kDa), rabbit anti-TrkB (92 kDa), phosphorylated-TrkB (91 kDa), AKT(56 kDa), phosphorylated-AKT (56 kDa, phospho S473), CREB (37 kDa), phosphorylated-CREB (37 kDa, phospho S133) (1:1000; Abcam), and mouse anti-α-tubulin (1:1000; Cell Signaling Technologies, 50 kDa) overnight at 4 °C after blocking with 5% skimmed milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at 25 °C. The membranes were then washed in TBST and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (1:10,000; Abcam). Immunoreactivity was visualized by using the enhanced chemiluminescence method.