Ab236632

Protein extracts were prepared and incubated with anti-bodies against HA or IgG for 24 h on a rotating wheel. Then, Pierce Protein A/G Magnetic Beads (ThermoFisher, USA) were added and incubated for another 24 h. After the beads were boiled, the precipitated proteins were separated by SDS-PAGE and transferred to PVDF membranes for further analysis. For western blotting, cell samples were extracted and quantified then boiled at 95 ℃, 10 min. Protein sample was separated on a 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel then transferred on a polyvinylidene fluoride (PVDF) membrane. Incubating primary antibodies overnight at 4 ℃, with specific primary antibodies against HA (1:1000, ab236632, Abcam), Cubilin (1:1000, ab191073, Abcam), anti-Amnionless antibody (1:500, sc-365384, Santa Cruz) and β-tubulin (AB0039, 1:2000, Abways) in Tris-Buffered Saline Tween-20 (TBST) containing 5% skim milk. After washed for 3 times with TBST, the membranes were incubated for 1 h at room temperature with a respective IgG-HRP labeled second antibody (1:10,000) in TBST containing 5% skim milk. Antigens were revealed using a chemiluminescence assay (Pierce ECL Western Blotting Substrate, 32,209, ThermoFisher, USA) and quantification of bands was achieved by densitometry using the Image J software.

Kidney biopsies and 293 T cells fixed in neutral buffered formalin were embedded in paraffin or optimal cutting temperature compound by using standard procedures. Frozen and paraffin sections were stained with immunofluorescence, respectively. Immunofluorescent staining and images were obtained by a Nikon A1R Meta confocal microscope. Cover slips were observed.
The antibodies used were list below: anti-Cubilin-C-terminal antibody (1:500, ab191073, Abcam), Rat Cubilin(CUBN) polyclonal antibody (1:100, 31010, Bicell Scientific), anti-Synaptop-odin antibody (1:50, 21064-1-AP, Proteintech), anti-Wilms Tumor Protein antibody (1:50, ab89901, Abcam), anti-COL4A3 antibody (1:100, Kingmed, Guangzhou, China), anti-COL4A5 antibody (1:100, Kingmed, Guangzhou, China), anti-Amnionless antibody (1:10, sc-365384, Santa Cruz), anti-Megalin Antibody (1:30, CD7D5, Novus Biologicals), goat polyclonal secondary antibody to mouse Alexa fluor 488 (1:400, ab150113, Abcam), goat polyclonal secondary antibody to rabbit Alexa fluor 555 (1:400, ab150078, Abcam), rabbit monoclonal to HA tag (1:500, ab236632, Abcam), goat polyclonal secondary antibody to rabbit Alexa fluor 647 (1: 400, ab150079, Abcam), goat anti-mouse Alexa fluor 568 (1: 400, ab175473, Abcam), DAPI (1:1000, C1002, Beyotime).

One day before transfection, HEK293T cells were seeded into a 6-well plate. For each well, 2 μg of Cas9-expressing plasmid were transfected using 4 μL of Lipofectamine2000. Three days after transfection, cell samples were collected and total proteins were extracted using NP-40 buffer (Beyotime) supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF) (Beyotime). The protein was separated by SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) (Thermo) membrane. After transfer, the membrane was blocked with 5% (wt./vol.) BSA (Sigma) in TBS-T (0.1% Tween 20 in 1× TBS) buffer and then incubated in the primary antibody (anti-HA tag (1:1,000; ab236632, Abcam) and anti-GAPDH (1:2,000; 5174s, Cell Signaling) at 4°C overnight. Wash membrane three times in TBS-T for 5 min each time. The second antibody (1:10,000; ab6721, Abcam) was incubated for 1 h at room temperature, and then washed three times and imaged.

Cells were lysed with RIPA buffer, separated with Bolt™ 4-12% Bis-Tris Plus Gels (Invitrogen) and transferred to nitrocellulose membranes. The membranes were blocked with 5% blotting-Grade Blocker (Cat # 1706404, Bio-RAD) using 1xTBST buffer (Cat # T9511, Teknova) for 1 h and incubated overnight at 4 °C with following primary antibodies: anti-HA antibody (1:1000, ab236632, Abcam), anti-GST antibody (1:1000, Cat # 8-326, Thermofisher), anti-CPSF6 antibody (1:5000, ab175237, Abcam), anti-Sec24C antibody (1:1000, ab122633, Abcam), anti-Nup153 antibody (1:2000, NB100-93329, Novus), anti-GAPDH antibody (1:3000, sc-47724, Santa Cruz). The membranes were subsequently incubated with a goat anti-rabbit IgG (H + L) secondary antibody (1:3000, 65-6120, Invitrogen) or goat anti-mouse IgG (H + L) secondary antibody conjugated (1:3000, 65-6520, Invitrogen) to horseradish peroxidase and visualized by enhanced chemi-luminescence (RPN2232, Cytiva).

This assay was used to detect whether CENPN could directly bind to the target protein CREB. GST-CENPN and HA-CREB genes were first cloned and synthesized and then inserted into pGEX-6P-1 (Sigma, GE28-9546-48). After insert ligation, transformation into recipient cells and clonal expression, the protein samples containing 500 μg GST (control group) or GST-CENPN (experimental group) were added into glutathione-agarose resin (Thermo Scientific, 16100) and mixed for 3 h, respectively. Five hundred micrograms of HA-CREB protein were added to the control and experimental groups and then mixed overnight. The two groups of samples were centrifuged, and an appropriate amount of protein loading buffer was added and then incubated at 100°C for 5 min. Finally, WB experiments were performed with anti-GST (abcam, ab111947) and anti-HA (abcam, ab236632) antibodies for GST: GST-CENPN and HA-CREB, respectively.

PLA experiments were conducted as described^{1 (link)}. HEK293T cells were seeded on coverslips in a 24-well dish. The cells were challenged with VSV-G pseudotyped HIV-1 (500 ng of p24), washed and supplied with fresh medium at 1 hpi. The cells were fixed with 4% paraformaldehyde for 15 min at 6 hpi and permeabilized with 0.1% Triton X-100 for 15 min at room temperature. After washing with a blocking buffer supplied by Duolink In Situ Red kit (DUO92101, Sigma–Aldrich) for 1 h at 37 °C, the cells were incubated with anti-HIV-1 p24 antibody AG 3.0 (ARP-4121, NIH AIDS Reagent Program) at 1:100 dilution and anti-HA antibody (ab236632, Abcam) (1:1000) for 1 h at room temperature. Samples were processed further using the Duolink In Situ Red kit according to the manufacturer’s instructions. Signals were detected by using an Olympus FV1000 laser scanning confocal microscope.

The following primary antibodies were used: anti-tubulin (Abcam, ab210797), anti-KMT5A (Abcam, ab111691), anti-CD8 (Abcam, ab17147), anti-CD8 (Abcam, ab217344), anti-CD3 (Abcam, ab16669), anti-ubiquitin (CST, #43124), anti-CD69 (Abcam, ab54217), anti-FLAG (Abcam, ab205606), anti-HA (Abcam, ab236632), anti-6X His (Abcam, ab213204), anti-CD73 (Abcam, ab54217), anti-CD73 (Abcam, ab54217), anti-COP1 (Abcam, ab56400), anti-MKRN1 (Abcam, ab72054), anti-MDM2 (BOSTER, BA3612–2), and anti-Ki-67 (CST, #12202), anti-CD69 (BOSTER, A00529–2), anti-IFN-γ (Abcam, ab231036), anti-IL-2 (Abcam, ab92381), anti-TNF-α (Abcam, ab270264), anti-perforin (Abcam, ab47225), anti-Granzyme B (BOSTER, A00353), anti-perforin (Abcam, ab16074). The following secondary antibodies were used: goat anti-mouse (CST) and goat anti-rabbit (CST). MG132 and cycloheximide (CHX) were purchased from CST (USA). The CMV peptide pool stimulating CD8⁺ T cells was obtained from Mabtech (Sweden).