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5 protocols using anti ubiquitin k48

1

Characterizing CRBN-mediated Protein Degradation

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The following antibodies were used: anti-FLAG (Sigma, F7425), anti-IKZF3 (Cell Signaling, #15103), anti-IKZF1 (Cell Signaling, #9034), anti-RUNX1 (Abcam, ab92336), anti-RUNX3 (Cell Signaling, #9647), anti-CBFβ (Santa Cruz, sc-20693), anti-CRBN (Sigma, HPA045910), anti-ubiquitin (K48) (EMD Millipore, #05–1307), anti-TUBULIN (Santa Cruz, sc-8035), anti-VINCULIN (Santa Cruz, sc-73614), anti-Rabbit IgG-HRP (GE Healthcare, NA934V), and anti-mouse IgG-HRP (GE Healthcare, NA931V). The following agarose beads were used: anti-FLAG M2 Affinity Gel (Sigma, A2220) and Glutathione Sepharose 4B (GE Healthcare, #17075601). The following in vitro translation kit was used: TNT T7 Coupled Reticulocyte Lysate Systems (Promega, L4610). Benzonase was used according to manufacturer (Sigma, E1014). The following compounds were used: Lenalidomide (Sigma, CDS022536), Pomalidomide (Sigma, P0018), Bortezomib (Millennium Pharmaceuticals), and AI-10–104 was kindly provided by Dr. John H. Bushweller.
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2

Immunoprecipitation Assay for E3 Ligase Complexes

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The following antibodies were used: anti-FLAG (Sigma, F7425), anti-IKZF3 (Cell Signaling, #15103), anti-IKZF1 (Cell Signaling, #9034), anti-RUNX1 (Abcam, ab92336), anti-RUNX3 (Cell Signaling, #9647), anti-CBFβ (Santa Cruz, sc-20693), anti-CRBN (Sigma, HPA045910), anti-ubiquitin (K48) (EMD Millipore, #05-1307), anti-TUBULIN (Santa Cruz, sc-8035), anti-VINCULIN (Santa Cruz, sc-73614), anti-Rabbit IgG-HRP (GE Healthcare, NA934V), and anti-mouse IgG-HRP (GE Healthcare, NA931V). The following agarose beads were used: anti-FLAG M2 Affinity Gel (Sigma, A2220) and Glutathione Sepharose 4B (GE Healthcare, #17075601). The following in vitro translation kit was used: TNT T7 Coupled Reticulocyte Lysate Systems (Promega, L4610). Benzonase was used according to manufacturer (Sigma, E1014). The following compounds were used: Lenalidomide (Sigma, CDS022536), Pomalidomide (Sigma, P0018), Bortezomib (Millennium Pharmaceuticals), and AI-10-104 was kindly provided by Dr. John H. Bushweller.
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3

Comprehensive Antibody Resource for Research

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The following antibodies have been described: anti-GFP (#GF200; Nacalai Tesque, Kyoto, Japan), anti-RFP (#R10367; Thermo Fisher Scientific), anti-caspase 3 (#9662; Cell Signaling Technology, Danvers, MA), an α-tubulin antibody (DM1A; Merck Millipore, Darmstadt, Germany), anti-TDP43 (#3448; Cell Signaling Technology), anti-phosphorylated TDP43 (#1D3; Merck Millipore), anti-Ubiquitin K48 (#Apu2; Merck Millipore), anti-FLAG (M2; Sigma-Aldrich, St. Louis, MO), horse radish peroxidase-conjugated anti-mouse or rabbit IgG (The Jackson Laboratory, Bar Harbor, ME), and AlexaFluor 488/594-conjugated anti-mouse, rabbit, or rat IgG antibody (Thermo Fisher Scientific).
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4

Antibody Immunoblotting for Cellular Signaling

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Antibodies against phosphorylated and total IRF3, IKKε, TBK1, NF-κB p65, ERK, JNK, and p38 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-TRAF3, anti-β-actin, and HRP-conjugated secondary antibodies were from Santa Cruz. Anti-Myc, anti-Flag, anti-HA, and anti-GFP were from Origene. Anti-PINK1, anti-Parkin, and anti-YAP1 were from Abcam Biotechnology (Cambridge, UK). Anti-K48-ubiquitin and anti-K63-ubiquitin were from Millipore (Kenilworth, USA).
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5

Investigating AKT Regulation in Cellular Processes

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Chemicals were obtained from the following sources: cis-Diammineplatinum (II) dichloride (CDDP), N-acetylcysteine (NAC) and MG132 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were obtained from the following sources: anti-AKT (#9272 for western blot), anti-AKT (#2920 for proximity ligation assay, PLA), anti-p-AKT (Ser473), anti-Myc, anti-His, anti-FOXO1, anti-FOXO3, anti-FOXO4, anti-GAPDH, anti-Normal Rabbit IgG, horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG (Cell signaling Technology, Beverly, MA, USA), anti-K48 Ubiquitin, anti-K63 Ubiquitin, anti-α-Tubulin (Millipore, Temecula, CA, USA), anti-HA for western blot, anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MUL1, anti-Lamin A and anti-HA for ChIP assay (abcam, Cambridge, MA, USA), anti-hemagglutinin (HA), anti-GFP (Santa Cruz, Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma-Aldrich), Alexa Flour 488-conjugated goat anti-Mouse and Alexa Flour 546-conjugated goat anti-Rabbit (Invitrogen, Carlsbad, CA, USA)
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