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4 protocols using mouse anti ha 16b12

1

Western Blot and Immunofluorescence Antibodies

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For western blot, the following antibodies were used: rabbit anti-Ago2 (Sigma) at 1∶1000, mouse anti-HA (16B12, Covance) at 1∶2000, mouse anti-Ubc9 (BD biosciences) at 1∶1000, mouse anti-tubulin (Sigma) at 1∶10 000, in house rabbit anti-SUMO1 at 1∶1000, mouse anti-RanBP2 (Santa Cruz) at 1∶400, rabbit anti-SAE2 (Abcam) at 1∶1000, mouse anti-Ago1 (Upstate) at 1∶1000, mouse anti-Vinculin (Abcam) at 1/1000, rabbit anti-PARP1 (Santa Cruz) at 1∶1000 and mouse anti-GFP (Sigma) at 1∶500. For immunofluorescence, antibodies used are the following: rabbit anti-Dcp2 (Sigma) at 1∶300, mouse anti-HA (16B12, Covance) at 1∶500, rabbit anti-Ago2 (Abcam) at 1∶300. DAPI was purchased from Invitrogen. Cycloheximide and NaAsO2 solution were purchased from Sigma.
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2

Immunofluorescence Staining Protocol

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Rabbit anti-CHC (ab21679), rabbit anti-GFP (ab290), goat anti-GFP (ab6673), rabbit anti-α-adaptin (ab189995), and rabbit anti-γ-adaptin (ab220251), as well as CytoPainter Phalloidin-iFluor 405 (ab176752), were purchased from Abcam. Mouse anti-β1 integrin (MEM-101A) was purchased from Novus Biologicals, mouse anti-HA (16B12) from Covance, and mouse anti-γ-adaptin (100/3) from Sigma. Hybridomas producing antibodies against LDL receptor (C7) were purchased from the American Type Culture Collection. Unlabeled, generic goat anti-rabbit IgG antibodies were purchased from Zymed. A polyclonal antibody cross-reacting between µ1A and µ1B was a kind gift from Linton Traub (University of Pittsburgh, Pittsburgh, PA).
Secondary antibodies labeled with Alexa dyes donkey anti-mouse Alexa 647, donkey anti-mouse Alexa 568, goat anti-mouse Alexa 488, donkey anti-goat Alexa 680, and donkey anti-goat Alexa 488 were purchased from Molecular Probes/Thermo Fisher Scientific. Donkey anti-mouse IRDye 800CW and donkey anti-rabbit IRDye 680RD antibodies were purchased from Li-Cor. Donkey anti-rabbit Cy5 antibodies, and peroxidase–conjugated goat anti-mouse and goat anti-rabbit antibodies were from Jackson ImmunoResearch. Goat anti-mouse antibodies labeled with 1.4-nm colloidal gold as well as HQ Silver enhancement kit were from Nanoprobes. DAPI solution was purchased from BD Biosciences.
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3

Standard SDS-PAGE Gel Analysis Protocol

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Standard SDS-PAGE gel analysis was carried out in a range of gel concentrations. Proteins were either stained with GelCode Blue (ThermoFisher) or transferred to PVDF Immobilon-P transfer membranes (0.45 µM pore size) (Sigma-Aldrich) for immunoblot analysis.35 (link) Antibodies utilized for immunoblotting were: mouse anti-tetraHis (Qiagen, 1:4,000); mouse anti-FLAG M2 (Sigma, 1:10,000); rabbit anti-ubiquitin (Dako, 1:1000); mouse 16B12 anti-HA (Covance, 1:1000); and mouse anti-PGK (yeast phosphoglycerate kinase) (Molecular Probes, 1:20,000). Secondary antibodies used were: sheep anti-mouse NA931V (GE Healthcare, 1:10,000) and donkey anti-rabbit NA934V (GE Healthcare, 1:5,000). Membranes used for anti-His blotting required blocking of nonspecific binding with 3% (w/v) BSA and extensive washing. Other immunoblot analyses used 5% milk for blocking.
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4

Standard SDS-PAGE Gel Analysis Protocol

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Standard SDS-PAGE gel analysis was carried out in a range of gel concentrations. Proteins were either stained with GelCode Blue (ThermoFisher) or transferred to PVDF Immobilon-P transfer membranes (0.45 µM pore size) (Sigma-Aldrich) for immunoblot analysis.35 (link) Antibodies utilized for immunoblotting were: mouse anti-tetraHis (Qiagen, 1:4,000); mouse anti-FLAG M2 (Sigma, 1:10,000); rabbit anti-ubiquitin (Dako, 1:1000); mouse 16B12 anti-HA (Covance, 1:1000); and mouse anti-PGK (yeast phosphoglycerate kinase) (Molecular Probes, 1:20,000). Secondary antibodies used were: sheep anti-mouse NA931V (GE Healthcare, 1:10,000) and donkey anti-rabbit NA934V (GE Healthcare, 1:5,000). Membranes used for anti-His blotting required blocking of nonspecific binding with 3% (w/v) BSA and extensive washing. Other immunoblot analyses used 5% milk for blocking.
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