Anti tau

Slice cultures were fixed with 4% paraformaldehyde overnight at 4 °C and incubated in blocking buffer (10% normal goat serum in 0.1% Triton-X in PBS) for 1 h. Cultures were left in primary antibody (1:200 anti-tau, abcam; cat. #ab75714), 1:500 anti-MAP2 (BD Pharmingen, San Diego, CA, USA; cat. #556320), 1:200 anti-DRP1 EPR19274 (abcam; cat. #ab184247), 1:200 anti-NCX1 EPR12739 (abcam; cat. #ab177952), and 1:200 anti-TOMM20 (abcam; cat. #ab56783) overnight at 4 °C, washed 3× with PBS, and incubated with respective secondary antibody conjugated to Alexa Fluor 488 or 647 (1:500; ThermoFisherScientific). Fluorescence micrographs were taken under a ×20 objective using the EVOS FL Microscope (ThermoFisherScientific). The following filter cubes were used for this study: EVOS™ Light Cube, GFP cat. AMEP4651; EVOS™ Light Cube, RFP cat. AMEP4652; EVOS™ Light Cube, Cy™5 cat. AMEP4656. The Look Up Table (LUT) applied to display CellROX Green fluorescence intensity is linear and found in ImageJ (16-color LUT). Colocalization of TOMM20 and Drp1 immunostaining was determined using the Coloc 2 plugin (https://imagej.net/Coloc_2) in ImageJ.

To assess centrosome numbers, MDA-MB-231, MDA-MB-468, SUM1315 or HCC1937 cells were fixed in 10% phosphate buffered formalin, permeabilized with methanol/acetone (1:1, v/v), and immunostained with anti-pericentrin (Abcam, MA) antibody and the corresponding FITC-conjugated secondary antibody (Molecular Probes, Eugene, OR). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). To evaluate the effects of SMI#9 and PTX treatments on TAU localization and MT spindles, MDA-MB-468 or HCC1937 cells were treated overnight with 0.75 µM SMI#9, PTX (2.5 nM, MDA-MB-468 cells; 10 nM, HCC1937 cells) or a combination of 0.75 µM SMI#9 and PTX, and subjected to immunostaining with anti-α-tubulin (Sigma-Aldrich Chemicals, MO) and anti-TAU (Abcam, MA) antibodies and the corresponding Texas Red or FITC-conjugated secondary antibodies, respectively. Nuclei were counterstained with DAPI and scored for TAU-positive cells and polarity of MT spindles. To assess nonspecific reactions, slides were stained in the absence of primary antibody or with isotype matched nonimmune IgG. Images were collected on an Olympus BX40 microscope equipped with a Sony high resolution/sensitivity CCD video camera and processed using CellSens software. The results shown are representative of data collected from at least 40 cells in five-seven fields and from two independent experiments.

Two hybridoma strains for the mouse anti-rat NogoA protein were preserved by the Institute of Neurosciences in the Fourth Military Medical University, and the mouse IgG was purified as described previously [15] (link). We purchased the following primary antibodies: polyclonal rabbit anti-NogoA antibody (pAb) (Alpha Diagnostic Intl., USA), rabbit anti-MBP mAb, rabbit anti-GFAP mAb (Denmark DAKO, USA), rabbit anti-GST (Sigma, USA), anti-Tau (Abcam, USA), anti-Map2 (Sigma, USA), anti-βIII-tubulin (Anbo, USA), and anti-β-actin (Anbo, USA). The following secondary antibodies were used: (FITC)-labelled goat anti-mouse immunoglobulin (IgG), Alexa-594-labelled goat anti-rabbit IgG (Abcam, USA), and hydrogen peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG (Jackson Immuno Research Company, USA). Recombinant Rat NogoA/Fc Chimera (aa 544–725) and Recombinant Rat NogoA/Fc Chimera (aa 1026–1090) were purchased from R&D Systems.

The following antibodies were used in this study: anti-Fyn (catalogue no. ab1881, Abcam), anti-Fyn (catalog no.: sc-434, Santa Cruz Biotechnology), anti-APP (catalog no.: ab32136, Abcam), anti-actin (catalog no.: TA-09, Zsgb-Bio, China), anti-NMDAR2B (catalog no.: ab254356, Abcam), anti-ZDHHC21 (catalog no.: PA5-25,096, Invitrogen, USA), anti-NeuN (catalog no.: ab279295, Abcam), anti-tau (phospho T231; catalog no.: ab151559, Abcam), anti-tau (phospho Thr181; catalog no.: 701530, Invitrogen), anti-Aβ (catalog no.: ab201060, Abcam), anti-phospho-Src family (catalog no.: 2101S, Cell Signaling Technology, USA), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG heavy chain-specific secondary antibody (catalog no.: A25222, Abbkine, China), HRP-conjugated mouse anti-rabbit IgG light chain (catalog no.: A25022, Abbkine), goat anti-mouse IgG H&L (HRP; catalog no.: ab6789, Abcam), goat anti-rabbit IgG H&L (HRP; catalog no.: ab205718, Abcam), goat anti-rabbit IgG H&L (Cy3; catalog no.: ab6939, Abcam), and goat anti-mouse IgG H&L (FITC; catalog no.: ab6785, Abcam).

Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM, Hyclone) supplemented with 1% penicillin/streptomycin (P/S) and 10% fetal bovine serum (Hyclone). Predesigned siRNA duplexes were purchased from Bioneer, and 10 nM siRNA transfection was carried out using RNAiMAX (Invitrogen, Waltham, MA, USA). Cells and tissues were lysed with N-PER™ and T-PER™ lysis buffer (Thermo Scientific, Waltham, MA, USA) supplemented with protease inhibitor cocktail (Roche, Hoffmann-La Roche AG, Basel, Switzerland), respectively. Lysates were resolved by SDS-PAGE gels and transferred onto nitrocellulose (NC) membranes (Merck Millipore, Burlington, MA, USA), and then antibodies were detected using the D-Plus ECL Femto System (Dongin Bio, Seoul, Korea) and the ImageQuant LAS4000 (GE Healthcare, Chicago, IL, USA). Antibodies used for immunoblotting included the following: anti-Ubiquitin (P4D1, Santa Cruz, Santa Cruz Biotechnology, Dallas, TX, USA), anti-UBE2H (18-Z, Santa Cruz), anti-Parkin (PRK8, Santa Cruz), anti-GAPDH (0411, Santa Cruz), anti-β-actin (C4, Santa Cruz), anti-Tau (Abcam, Cambridge, UK), and anti-UBE2L6 (MyBioSource, San Diego, CA, USA).