SHARPIN Antibody - BSA Free

Western Blot: SHARPIN Antibody - BSA Free [NBP2-04116] -

Linear ubiquitin chain assembly complex (LUBAC) assembles heterotypic poly-ubiquitin chains containing M1 and non-Lys linkages in vitro.(A) Time course of co-purified LUBAC in vitro ubiquitin chain assembly reaction. (B) Comparison of in vitro chain assembly between HOIL-1-interacting protein (HOIP), heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L), and Shank-associated RH domain-interacting protein (SHARPIN) mixed at 1:1:1 molar ratio versus co-purified LUBAC. (C) LUBAC in vitro chain assembly using different ubiquitin K to R mutants. (D) LUBAC in vitro chain assembly using K0 ubiquitin. (E) Ubiquitin chain restriction (UbiCRest) analysis of poly-ubiquitin chains assembled by LUBAC in vitro. All experiments were performed in triplicate representative results are shown.Anti-linear ubiquitin antibody validation.(A) Detection of mono-ubiquitin and di-ubiquitin of different linkages by anti-linear ubiquitin antibody. (B) Detection of longer Lys48 and Lys63-linked ubiquitin chains by anti-linear ubiquitin antibody.Independently purified HOIL-1-interacting protein (HOIP), heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L), and Shank-associated RH domain-interacting protein (SHARPIN) mixed at an equimolar ratio cannot reconstitute the trimeric linear ubiquitin chain assembly complex (LUBAC).Independent mass photometry (MP) measurements and weighted average made from HOIP, HOIL-1L, and SHARPIN mixed to a final concentration of 4 pM of each protein. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34142657), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: SHARPIN Antibody - BSA Free [NBP2-04116] -

Generation of Hoil-1lC458A/C458A mice.(A) Sequences of genomic DNA around C458 codon, gRNAs, and donor oligonucleotide used to target heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L) C458A mutation. (B) Sanger sequencing confirming correct mutations at target sites. (C) Genotyping results of Hoil-1l+/+, Hoil-1l+/C458A, and Hoil-1lC458A/C458A mice. Hpy188III digest of a PCR fragment confirming correct targeting where a silent mutation is inserted. (D) Immunoblot analysis of linear ubiquitin chain assembly complex (LUBAC) component expression in mouse embryonic fibroblasts (MEFs) derived from Hoil-1l+/+ and Hoil-1lC458A/C458A mice. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34142657), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Western Blot: SHARPIN Antibody - BSA Free [NBP2-04116] -

GFP-tagged LUBAC purification.(A) Purification of LUBAC containing GFP-tagged HOIP or HOIL-1L. (B) Mass photometry measurement of GFP-tagged LUBAC showing 27 kDa shift in the monomeric (250kDa) and dimeric (500kDa) mass of the complex. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/34142657), licensed under a CC-BY license. Not internally tested by Novus Biologicals.