Cells or tissues were lysed in ice-cold RIPA buffer (50 mM Tris, 150 mM NaCl, 1.0% Triton, 1 mM EDTA, 0.1% SDS) supplemented with protease (CAT #11697498001, Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (CAT #4693132001, Roche, Basel, Switzerland). The protein concentrations were determined by the BCA Assay using BCA Assay protein Kit (Sangon, Shanghai, China). Soluble protein was separated by 10% SDS-PAGE and transfer onto PVDF membranes (Millipore, Billerica, MA, USA). The membrane was incubated in blocking buffer (1xPBST with 1% BSA) for 1 h at room temperature. The membrane was then blotted with the diluted primary antibodies: anti-CPSF6 (1:1000, CAT #ab175237, Abcam), anti-NQO1 (1:1000, CAT #11451–1-AP, Proteintech) and anti-β-actin (1:5000, CAT #A5316, Sigma). Immunoreactive protein was visualized using ECL start Western Blotting Substrate (GE Healthcare Life Sciences, USA) and image quant LAS 4000 mini (GE Healthcare Life Sciences, USA).
Ab175237
Manufactured by Abcam
Sourced in Japan
Ab175237 is a protein-based detection reagent. It is designed to be used in immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab122633, by Abcam (3 mentions)
Ab133616, by Abcam (2 mentions)
Ab24700, by Abcam (2 mentions)
Ab109709, by Abcam (2 mentions)
Sc 32233, by Santa Cruz Biotechnology (2 mentions)
Ecl start western blotting substrate, by GE Healthcare (1 mentions)
A5316, by Merck Group (1 mentions)
Las 4000 mini, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Protease, by Roche (1 mentions)
Ab31631, by Abcam (1 mentions)
Anti rabbitigg h l alexa fluor 555 conjugate, by Cell Signaling Technology (1 mentions)
V 5507, by Merck Group (1 mentions)
Sc 33641, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg hrp, by Huabio (1 mentions)
Goat anti rabbit igg hrp, by Huabio (1 mentions)
Ab236632, by Abcam (1 mentions)
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Sc 47724, by Santa Cruz Biotechnology (1 mentions)
7 protocols using ab175237
1
Western Blot Analysis of Protein Expression
2
Innate Immune Response Signaling Pathway
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Anti-CPSF5 (1:5000; 10322-1-AP, Proteintech), anti-CPSF6 (1:5000; ab175237, Abcam), anti-CPSF7 (1:2000; 55195-1-AP, Proteintech), anti-RIG-I (1:1000; D14G6, Cell Signaling), anti-MDA5 (1:1000; D74E4, Cell Signaling), anti-IFIT3 (1:2000; 15201-1-AP, Proteintech), anti-ISG15 (1:2000; 15981-1-AP, Proteintech), anti-DDX21 (1:2000; 10528-1-AP, Proteintech), anti-DHX15 (1:2000; 12265-1-AP, Proteintech), anti-CCL2 (1:2000; 66272-1-Ig, Proteintech), anti-IRF3 (1:1,000; sc-33641, Santa Cruz), anti-phosphorylated IRF3 (1:1000; 4D4G, Cell Signaling), anti-TBK1 (1:1000; D1B4, Cell Signaling), anti-phosphorylated TBK1 (1:1000; D52C2, Cell Signaling), anti-VSVG (1:1000; V5507, Sigma), anti-ICP27 (1:1000; ab31631, Abcam), anti-GAPDH (1:20000; 60004-1-Ig, Proteintech), anti-HA (1:5000; clone HA-7, H6533, Sigma), anti-Flag (1:5000; clone M2, F1804, Sigma), Anti-rabbit IgG (H+L), (Alexa Fluor 555 Conjugate) (1:1000, 4413, Cell Signaling), goat anti-mouse IgG-HRP (1:10000; HA1006, HuaBio), goat anti-rabbit IgG-HRP (1:10000; HA1001, HuaBio). The poly(I:C) (LMW) and poly(dA:dT) were purchased from InvivoGen. Lipofectamine 3000 and Lipofectamine RNAiMAX were purchased from Invitrogen. Act D (actinomycin D) was purchased from Sigma.
3
Western Blot Analysis of Cellular Proteins
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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).
4
Inducible MxB Cell Lines for Virus Research
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Cell lines were cultured in Dulbecco’s Modified Eagle’s Media (DMEM) (Gibco/BRL; Paisley, UK) supplemented with 10% (v/v) fetal calf serum (FCS) and penicillin (Pen; 100 U/ml), streptomycin (Strep; 100 μg/ml) unless otherwise stated. G418 was used at 1000 μg/ml. Puromycin was used at 5 μg/ml. 293 cell lines were incubated at 37°C, 5% (v/v) CO2. All other cell lines were incubated at 37°C, 10% (v/v) CO2. Inducible MxB cell lines were made by transduction of HEK293, THP-1 and A3.01 with pLVX-Tet3G (Clontech), containing the Tet-responsive transactivator, and cells were selected for one week in G418. Cells were subsequently transduced with pLVX-mCherryKilled-MxB (modified from Clontech) and selected for one week in puromycin (2 μg/ml). Single cell clones were produced by limiting dilution in 96 well plates and assayed for optimal MxB induction. MxB inducible cell lines were induced for MxB expression by addition of 2 μg/ml of doxycycline for 48 hr prior to infection. Cell lines used in this study HEK293, U87-MG, HeLa, THP-1, A3.01. Cells routinely tested negative for mycoplasma using the Lonza MycoAlert mycoplasma detection kit. Antibodies used in this study; TNPO3 (ab54353, Abcam), CPSF6 (ab175237, Abcam), MxB (sc47197 N-17, Santa Cruz Biotechnology), CypA (BML-SA296-0100, ENZO), Nup358 (C288), Tubulin (DM1A, EMD Millipore), β-actin (ab6276, Abcam).
5
Antibody Detection of Trafficking Proteins
6
Antibody Detection of Trafficking Proteins
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The following antibodies were used to detect Sec24C (ab122633, Abcam) (1:1000 dilution), Sec24D (A304–813A, Bethyl) (1:1000 dilution), CPSF6 (ab175237, Abcam) (1:1000 dilution), Nup153 (ab24700, Abcam) (1:1000 dilution), Trim5α (ab109709, Abcam)(1:1000 dilution), CD4 (ab133616, Abcam) (1:1000 dilution), Clathrin Heavy Chain (610499, BD Laboratories) (1:1000) and GAPDH (sc-32233, SantaCruz) (1:3000 dilution).
7
Western Blot Analysis of Serum Proteins
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Serum samples were separated through SDS-PAGE and were then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After blocking with Block Ace reagent (DS Pharma Biomedical, Osaka, Japan), membranes were incubated with goat polyclonal anti-SNX5 (1:2000, ab5983, abcam, Tokyo, Japan), rabbit polyclonal anti-SRFBP1 (1:2000, ab109598, abcam), rabbit polyclonal anti-DDB1 (1:2000, ab97522, abcam), rabbit monoclonal anti-CPSF6 (1:2000, ab175237, abcam), or mouse monoclonal anti-ACACA (1:2000, ab205883, abcam) antibody. The second western blot was conducted with rabbit polyclonal anti-SNX5 (1:2000, SAB2102260, Sigma-Aldrich, Tokyo, Japan) antibody. Immunoreactive bands were detected using enhanced chemiluminescence (EMD Millipore, Temecula, CA, USA) after incubation with horseradish peroxidase labeled anti-mouse, rabbit, or goat IgG (1:5000, Vector Laboratories, Burlingame, CA, USA). Signals were detected using C-DiGit Blot Scanner (LI-COR) and then band density was assessed with Image Studio DiGit software (version 5.2) [38 (link)]. The whole blot can be found at supplementary materials (Figures S2 and S3 ).
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