The cultured N2a cells fixed in 4 % paraformaldehyde were thoroughly rinsed with PBS three times and treated with 10 % Triton X-100 for 10 min to rupture the cell membranes. The fixed cells were then blocked with 10 % donkey serum. One hour later, the coverslips were incubated overnight at 4 °C with the following primary antibodies: anti-HA (sc-7392, 1:200, Santa Cruz Biotechnology) or anti-annexin A1 (sc-12740, 1:200, Santa Cruz Biotechnology). After washing three times, the fixed coverslips were incubated with Alexa Fluor 488-conjugated AffiniPure goat anti-mouse IgG (H+L) (Jackson Immuno Research, West Grove, PA) for 60 min at room temperature. Finally, the fluorescence was captured by fluorescence microscopy (IX73, Olympus). Regional fluorescence intensities were quantified using Image Pro Plus software. To measure regional intensities, small circles within the cytoplasmic or nuclear regions of each cell were selected using the elliptical marquee tool. The intensity within each circle was obtained using the histogram function for each color channel, which was selected using the layers/channels palette. The values were recorded, for evaluation of the ratio of the nuclear to cytoplasmic intensity; we examined up to 10 independent fields of cells and scored at least 100 cells for each measurement.
Anti ha sc 7392
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
Anti-HA (sc-7392) is a mouse monoclonal antibody specific for the hemagglutinin (HA) tag. The antibody is designed for the detection of HA-tagged recombinant proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag f3165, by Merck Group (3 mentions)
Anti gfp sc 9996, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh 60004 1 ig, by Proteintech (2 mentions)
Hrp conjugated anti mouse igg m8770, by Merck Group (2 mentions)
Alexa fluor 488 conjugated affinipure goat anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Anti gapdh 5174, by Cell Signaling Technology (1 mentions)
Pertussis toxin, by Enzo Life Sciences (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Anti plcγ1 sc 81, by Santa Cruz Biotechnology (1 mentions)
Anti flag 2368, by Cell Signaling Technology (1 mentions)
Anti gst 2622, by Cell Signaling Technology (1 mentions)
Anti flag m2 beads a2220, by Merck Group (1 mentions)
Cdcl2, by Sangon (1 mentions)
Anti β actin a5316, by Merck Group (1 mentions)
Anti flag f7425, by Merck Group (1 mentions)
Anti p53 fl393, by Santa Cruz Biotechnology (1 mentions)
Anti cleaved caspase 3 d175, by Cell Signaling Technology (1 mentions)
Malp 2, by Enzo Life Sciences (1 mentions)
Anti actin a2228, by Merck Group (1 mentions)
21 protocols using anti ha sc 7392
1
Immunofluorescence Imaging of HA and Annexin A1
2
Antibody and Chemical Reagent Protocol
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The anti-TRPC3/6/7 (sc-15058, 1/1,000), anti-GFP (sc-9996, 1/1,000), anti-AT1R (sc-1173, 1/1,000), anti-HA (sc-7392, 1/1,000), anti-PLCγ1 (sc-81, 1/1,000) antibodies were purchased from Santa Cruz. The anti-Flag (2368, 1/1,000), anti-GAPDH (5174, 1/1,000), anti-GST (2622, 1/1,000) antibodies were from Cell Signaling. The anti-HA-tag beads and anti-GFP-tag beads were from Medical & Biological Laboratories Co., Ltd. (Japan). The anti-Flag M2 beads (A2220) were from Sigma. The anti-β-arrestin-1 (A1CT, 1/5,000) and anti-β-arrestin-2 antibodies (A2CT, 1/2,000) were generous gifts from Dr R.J. Lefkowitz (Duke University). The pertussis toxin was from Enzo Life Sciences. The SNX482 was from Abcam. The LaCl3 and CdCl2 were purchased from Sangon Biotech (Shanghai) Co. The Fura-2-AM was from Invitrogen. The DMEM medium was from Thermo Scientific. All other chemical or reagents were purchased from Sigma.
3
Western Blot Analysis of Protein Levels
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Standard western blot assays were used to analyze the levels of protein [14 , 37 (link)]. Anti-p53 (FL393, 1:2 000 dilution, Santa Cruz), anti-MDM2 (2A10, 1:1 000), anti-Flag (F7425,1:10 000 dilution, Sigma), anti-BAG5(ARP61996-P050, 1:1 000 dilution, Aviva Systems Biology, San Diego, CA, USA), anti-HA (sc-7392, 1:2 000 dilution, Santa Cruz), anti-CHIP (sc-66830, 1:1 000 dilution, Santa Cruz), anti-cleaved-Caspase 3 (D175, 1:1 000 dilution, Cell Signaling, Danvers, MA, USA) and anti-β-actin(A5316, 1:20 000 dilution, Sigma) antibodies were used to determine the levels of p53, MDM2, Flag-BAG5, BAG5, HA-BAG5, CHIP, cleaved caspase 3 and β-actin, respectively.
4
Immune Signaling Pathway Reagents
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LPS (Escherichia coli, serotype 055:B5) and anti‐Flag (F3165), anti‐Myc (M4439), and anti‐actin (A2228) were from Sigma (St. Louis, MO, USA). Malp2, poly (I:C), flagellin, R‐848, and CpG DNA were obtained from Enzo Life Sciences (Farmingdale, NY, USA). Antibody to TBK1, phosphorylated TBK1, IRF3, and phosphorylated IRF3 were from Cell Signaling (Beverly, MA, USA); and anti‐HA (sc‐7392) was purchased from Santa Cruz (Dallas, TX, USA).
5
Molecular constructs of Mertk and Tim-4
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All plasmids made in this study were generated by a cloning strategy based on PCR and sequenced to confirm their accuracy of sequence. Mouse Mertk cDNA were purchased from Open Biosystem (Cat #: OMM5896-202524955). HA-Tim-4, Tim-4ECR-FLAG, Tim-4IgV, and Tim-4mucin were previously used [18 (link)]. MertkECR, MertkIg, and MertkFnIII constructs contain residues 19-497, 19-277, and 278-497, respectively. The antibodies used in this study were anti-FLAG (F1804, Sigma Aldrich, St. Louis, MO, USA), anti-HA (SC-7392, Santa Cruz biotechnology, Dallas, TX, USA), anti-HA (#3724, Cell signaling technology, Danvers, MA, USA), anti-GFP (ab290, Abcam, Cambridge, MA, USA), anti-GST (SC-138, Santa Cruz biotechnology, Dallas, TX, USA), anti-mouse Mertk (AF591, R&D Systems, Minneapolis, MN, USA), anti-Tim-4 (SC-79143, Santa Cruz biotechnology, Dallas, TX, USA), anti-Tim-4 (ab176486, Abcam, Cambridge, MA, USA), anti-Actin (SC-47778, Santa Cruz biotechnology, Dallas, TX, USA), and normal goat IgG control (AB-108-C, R&D Systems, Minneapolis, MN, USA). Fluorochrome-conjugated donkey anti-goat secondary antibody (Alexa Fluor 488, A-11055) and goat anti-rabbit secondary antibody (Alexa Fluor 594, A-11037, Alexa Fluor 405, A-31556) were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA).
6
Comprehensive Western Blot Antibody Panel
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Primary antibody: anti-β-actin (sc-47778, 1:1000), anti-HA (sc-7392, 1:1000) and anti-GFAP (sc-33673, 1:200) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); Anti-iNOS (18,985–1-AP, 1:500) was purchased from Proteintech Group (Wuhan, China); anti-CD16/32 (AF1460, 1:500) was purchased from R&D systems (Minneapolis, MN, USA); anti-NF-κB p65 (#8242, 1:1000), anti-IKKα (#11,930, 1:1000), anti-Phospho-NF-κB p65 (#3033, 1:1000), anti-Phospho-IKKα/β (#2697, 1:1000), anti-Phospho-IκBα (#2859, 1:1000), anti-IκBα (#4814, 1:1000), anti-NBR1(#9891, 1:1000), anti-cleaved PARP (5625, 1:1000), anti-cleaved caspase-3 (9664, 1:1000) and anti-cleaved caspase-9 (#20,750, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA); anti-SENP6 (HPA024376, 1:1000) were purchased from Sigma–Aldrich (St. Louis, MO, USA); anti-Iba1 (ab283319, 1:100) was obtained from Abcam (Boston, MA, USA) and anti-NeuN (MAB377, 1:200) was purchased from Millipore Biotechnology (Schwalbach, Germany). Protein A + G agarose beads were obtained from Beyotime Biotechnology (Shanghai, China), and Ni2+-NTA agarose was purchased from QIAGEN (Dusseldorf, Germany). CHX was obtained from Calbiochem (508,739; Darmstadt, Germany).
7
Immunoprecipitation and Western Blot Analysis
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Cells were lysed by EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, and 0.5% NP-40) containing protease inhibitor (Cocktail, 11697498001, Sigma) and phosphatase inhibitor (Calbiochem, P8139, Sigma) on ice for 10 min and centrifuged at 15,000×g at 4°C for 15 min with the supernatant collected. A total of 20 µL sample was selected as input, and the remaining was subjected to reaction with 10 µL Protein G magnetic beads, followed by overnight incubation with 1 µL anti-HA (sc-7392, 1/1000; Santa Cruz) or anti-PD-L1 (Cat # NBP1-76769, 1/1000; Novus Biologicals) at 4°C. After centrifugation at 5000×g for 5 min at 4°C, the supernatant was discarded, and the recovered complex was washed with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP-40) four times, separated by SDS-PAGE and subjected to subsequent western blot analysis with corresponding antibodies.
8
Subcellular Localization of HA-RPL11 under miR-101-3p Overexpression
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The U2OS cells were co-transfected with HA-tagged RPL11 plasmid and miR-101-3p mimic using Lipofectamine 2000TM reagent. After incubation for 48 h, the cells were fixed with 100% ice-cold methanol for 10 min, and then permeabilized in PBS containing 0.25% Triton X-100 for 10 min. After washing, the cells were stained with anti-HA (sc-7392, Santa Cruz) overnight at 4 °C, followed by Alexa Fluor 488-conjugated goat anti-mouse (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. The nucleolus was stained with anti-fibrillarin (ab5821, Abcam), and DNA was detected by DAPI (Sigma-Aldrich). The fluorescence response HA-RPL11 and fibrillarin staining cells were visualized using the Nikon ECLIPS Ti-U fluorescence microscope (Nikon, Tokyo, Japan) and analyzed using NIS-Elements imaging software (Nikon, Tokyo, Japan, ver 4.20).
9
Western Blot Antibody Reagents
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Anti-S6K (#9202), anti-phospho S6K Thr389 (#9234), anti-AKT (#9272), anti-phospho AKT Ser473 (#9271), anti-LAT1 (#5347), anti-mTOR (#2983), anti-Raptor (#2280), anti-Flag (#14793), and anti-Rictor (#2140) were purchased from Cell Signaling Technology. Anti-tubulin (05-829) was purchased from Millipore. Anti-β-actin (A700-057) and anti-Flag M2 (F1804) were purchased from Sigma. HA-horseradish peroxidase (HRP) (11-814-150-001) was purchased from Roche. Anti-HA (sc-7392) was purchased from Santa Cruz Biotechnology. Anti-LAMP2 (ab25631) and anti-sodium potassium ATPase (ab76020) were purchased from Abcam.
10
Yeast Protein Tagging and Western Blot Analysis
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All media components were either from BD or GIBCO, general reagents from Sigma Aldrich and Roche unless otherwise stated. Yeast strains were from a w303 isogenic background. All strains are listed in Table 1 . Gene disruption and protein tagging were performed as previously described (Janke et al. 2004 (link)) and controlled by PCR amplification and western blot assays. Growth media were: YPD (1% yeast extract, 2% bactopeptone, 2% glucose), SD (0.67% YNB and 2% glucose) and SC (0.67% YNB, 2% glucose or galactose, selective Drop-Out Mix). Benomyl was used at a final concentration of 10/15μγρ/μl. Antibodies were: anti-Myc (clone 9E10, sc-40, Santa Cruz), anti-ADA2 (sc-6651, Santa Cruz), anti-HA (sc-7392, Santa Cruz), and Anti-6X His tag (ab1187, Abcam).
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