Ab6161

Protein extracts were separated on 7.5 or 10% Mini-PROTEIN TGX Precast Gels (Bio-Rad) and then transferred onto nitrocellulose membranes with the Trans-Blot Turbo Transfer System (Bio-Rad). The following primary antibodies and dilutions were used: rabbit anti-PRDM9 polyclonal (Grey et al. 2017 (link)), 1:500; rabbit anti-CTCF monoclonal (Cell Signaling Technology, D31H2), 1:2000; rabbit anti-CXXC1 polyclonal (Millipore, ABE211), 1:5000; rat anti-tubulin α monoclonal (Abcam, ab6161), 1:3000; rabbit anti-PIH1D1 polyclonal (Proteintech, AP19427), 1:5000; rabbit anti-CEP70 polyclonal (Abnova, PAB17658), 1:1000; mouse anti-MCRS1 polyclonal (Abnova, H00010445-B01P), 1:250; anti-Gal4 AD (Millipore, 06-283), 1:3000; and anti-Gal4 BD (Sigma, G3042), 1:2000. Signals were detected with the horseradish peroxidase (HRP)-conjugated secondary antibodies, donkey anti-rabbit IgG HRP (Jackson ImmunoResearch Laboratories), goat anti-rat IgG HRP (GE Healthcare), and sheep anti-mouse IgG HRP (GE Healthcare), and then visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). ImageJ 1.49m was used to quantify signal intensities of western blot images captured by ChemiDoc™ XRS+ Imager with the Image Lab 5.1 software.

Detection of TAP tag was performed using an HRP-coupled antibody against protein A (A01435-100 Genscript). Detection of Utp21p, Pwp2p, and Utp18p was monitored using polyclonal primary antibodies generated against the untagged Pwp2p (1–716), untagged Utp21p (1–698), and untagged Utp18p by Agro-Bio, respectively. HRP-coupled anti-rabbit secondary antibody (111-035-003, Dianova) was used to detect polyclonal antibodies. Detection of Tubulin was performed with the monoclonal antibody YOL1/34 (ab6161, Abcam) and the HRP-coupled anti-rat secondary antibody (112-035-068, Dianova). Protein signals were visualized using the Chemiluminescence Western blotting reagent (Roche) in LAS-3000 (Fujiflm).

Cell culture reagents were purchased from Life Technologies (Grand Island, NY). HEK-293 cells were purchased from ATCC (#CRL-1573). The authentication of this cell lines was provided by ATCC, and the cell line was tested negative for mycoplasma contamination. Antibodies to DCX (ab18723), DHC (ab6305), DIC (ab23905), JIP3 (ab196761), and Tubulin (ab6161) were purchased from Abcam (Cambridge, MA). Antibody to HA was from EMD Millipore (Billerica, MA). Construct expressing IC-1B was generously provided by Dr. Kevin Pfister (UVA). Construct expression JIP3 was generously provided by Dr. Roger Davis (UMASS MED). Construct expression TrkB-RFP was generously provided by Dr. Xiaowei Zhuang (Harvard University). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Tissues were homogenized (Ultra-Turrax TP, IKA Labortechnik, Wasserburg, Germany) on ice in 300 µl of RIPA lysis buffer (Thermo) per 100 mg of tissue with 1× protease inhibitor cocktail (Thermo) and incubated for 30 min. Lysates were centrifuged at 14 000g, 4°C for 20 min and overall protein concentrations of the supernatant was determined by Pierce BCA Protein Assay (Life Technologies). Samples were incubated at 37°C for 30 min in 1× LDS sample buffer (Life Technologies) and 1× sample reducing agent (Life Technologies), after which 40 µg of protein were loaded per well in a NuPAGE Bis–Tris 4–12% polyacrylamide gel for protein separation via SDS-PAGE electrophoresis. Proteins were transferred to PDVF membrane at 400 mA for 1 h and membrane was blocked for 1 h at 4°C with 5% BSA in TBS + 3% Tween 20. Membranes were subsequently incubated overnight at 4°C with primary antibodies for NPC1 (1:10 000, ab134113, Abcam) and β-tubulin (1:2000, ab6161, Abcam) with 3% BSA in TBS + 3% Tween 20. After 3 washes in TBS, antibody staining was revealed using HRP-conjugated goat anti-rabbit IgG (1:2000, ab6721, Abcam) and goat anti-rat IgG (1:10 000, ab97057, Abcam) incubated for 2 h at RT in TBS + 3% Tween 20 with 3% BSA. Blots were developed with ECL system (SuperSignal West Pico, Life Technologies) and imaged using a Genegnome imager (Syngene, Cambridge, UK).

For the analysis of Cox2 protein expression, cells were harvested in logarithmic phase and lysed in 1.85 NaOH/7.4% ß-mercaptoethanol (39 (link)). Proteins were precipitated with 25% TCA, washed with acetone and resuspended in 5% SDS. After SDS-PAGE, proteins were transferred to nitrocellulose membranes, according to standard techniques. Membranes were incubated with 1:3000 anti-Cox2 antibody (40 (link)) followed by 1:50 000 anti-rabbit HRP antibody (Sigma, A0545), or with 1:2000 anti-tubulin antibody (Abcam, ab6161) followed by 1:50 000 anti-rat HRP antibody (Abcam, ab6734).
See Supplementary Information for further Materials and Methods.

Immunoblotting was performed with total cellular extract using standard protocols. Antibodies used were rabbit anti-53BP1 (Santa-Cruz, sc-22760) and rat anti-tubulin (Abcam, ab6161).

The methods of western blot analysis were described previously (28 (link)). Briefly, HTR-8/SVneo cells transfected with miRNAs were washed by PBS and lysed by RIPA lysis buffer. Samples consisting of 50 µg total protein were loaded onto an SDS-PAGE gel (P0012AC, Beyotime Biotechnology, China) and transferred electrophoretically to nitrocellulose membranes (LC2000, Invitrogen). After blocking with Tris-buffered saline with Tween-20 (TBST) containing 5% milk powder, the membranes were incubated with the appropriate primary antibody against HLA-G (MEM-G/1, ab7759; Abcam, Cambridge, UK, 1:500) or tubulin (ab6161, Abcam, Cambridge, UK, 1:2,000), anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody (GE Healthcare, Piscataway, NJ, USA) was added for 1 h.
The blots were developed using Immobilon Western HRP Substrate Luminol Reagent (Millipore, Billerica, MA, USA). The quantification of HLA-G relative to tubulin expression within each sample was determined using the QuantiOne imaging software (Bio-Rad, USA).