Human/Mouse/Rat p27/Kip1 Antibody

Detection of p27/Kip1 by Western Blot

In vitro ubiquitination of p27 in yeast extract. In vitro ubiquitination of p27 was carried out with yeast extracts with the indicated mutations, UbcH7, and His6-p27. His6-p27 was pulled down with Ni-NTA beads, and gels were blotted with anti-ubiquitin and anti-p27 antibodies. A, wild-type yeast extract shows strong ubiquitination of p27 (lane 1) whereas the no p27 (lane 3) and no extract (lane 2) controls show little ubiquitination. The addition of rsp5-1 mutant extract does not promote p27 ubiquitination (lane 4), even with the addition of excess extract (lane 6). The lower panel shows equal loading of p27 in lanes 1, 2 4, and 6. B, as in A, WT but not rsp5-1 mutant extract supports p27 ubiquitination (lane 1 vs lane 4). The addition of purified Rsp5 promotes p27 ubiquitination in a dose-dependent manner (lanes 4, 5, 6), while the addition of a catalytically dead mutant Rsp5 does not support p27 ubiquitination (lane 7). The lower panel shows p27 loading. The bottom panel shows GAPDH loading control. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38301893), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of p27/Kip1 by Western Blot

In vitro ubiquitination of p27 in yeast extract. In vitro ubiquitination of p27 was carried out with yeast extracts with the indicated mutations, UbcH7, and His6-p27. His6-p27 was pulled down with Ni-NTA beads, and gels were blotted with anti-ubiquitin and anti-p27 antibodies. A, wild-type yeast extract shows strong ubiquitination of p27 (lane 1) whereas the no p27 (lane 3) and no extract (lane 2) controls show little ubiquitination. The addition of rsp5-1 mutant extract does not promote p27 ubiquitination (lane 4), even with the addition of excess extract (lane 6). The lower panel shows equal loading of p27 in lanes 1, 2 4, and 6. B, as in A, WT but not rsp5-1 mutant extract supports p27 ubiquitination (lane 1 vs lane 4). The addition of purified Rsp5 promotes p27 ubiquitination in a dose-dependent manner (lanes 4, 5, 6), while the addition of a catalytically dead mutant Rsp5 does not support p27 ubiquitination (lane 7). The lower panel shows p27 loading. The bottom panel shows GAPDH loading control. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38301893), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of p27/Kip1 by Western Blot

UbcH7-SMURF1 prefers conjugating K29 ubiquitin to p27. Ubiquitination of p27 was carried out in a reconstituted system with Ube1, UbcH7, SMURF1, His6-p27, ATP regeneration system, and buffer. Reactions were run on SDS-PAGE gels and probed with anti-SMURF1 and anti-p27 antibodies. A, the p27 immunoblot shows p27 is ubiquitinated by SMURF1 with wild-type (lane 4 vs. lane 2) and K29O ubiquitin (lane 5 vs. lane 2) but not K29R (lane 6) or K63O ubiquitin (lane 7). B, the immunoblot for SMURF1 shows SMURF1 auto-ubiquitination with wild-type (lanes 3 and 4), K29O (lane 5), and, to a lesser extent, K63O ubiquitin (lane 7). Autoubiquitination is more extensive with p27 present (Lane 4 versus 3). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38301893), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of p27/Kip1 by Western Blot

UbcH7-SMURF1 prefers conjugating K29 ubiquitin to p27. Ubiquitination of p27 was carried out in a reconstituted system with Ube1, UbcH7, SMURF1, His6-p27, ATP regeneration system, and buffer. Reactions were run on SDS-PAGE gels and probed with anti-SMURF1 and anti-p27 antibodies. A, the p27 immunoblot shows p27 is ubiquitinated by SMURF1 with wild-type (lane 4 vs. lane 2) and K29O ubiquitin (lane 5 vs. lane 2) but not K29R (lane 6) or K63O ubiquitin (lane 7). B, the immunoblot for SMURF1 shows SMURF1 auto-ubiquitination with wild-type (lanes 3 and 4), K29O (lane 5), and, to a lesser extent, K63O ubiquitin (lane 7). Autoubiquitination is more extensive with p27 present (Lane 4 versus 3). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38301893), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of p27/Kip1 by Western Blot

A, His-p27 and FLAG-SMURF1 were allowed to interact, and then the p27 was pulled down with Ni-NTA beads. The flowthrough and bead bound proteins were analyzed by Western blot. His-p27 was able to pull down SMURF1. B, Myc-SMURF1 -HECT was immunoprecipitated from HELA cells using anti-Myc antibody and protein G beads. His-p27 was then incubated with the beads, and Western blots were used to analyze the bead bound proteins after washing. His-p27 did not interact with the truncated SMURF1. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38301893), licensed under a CC-BY license. Not internally tested by R&D Systems.