Anti-MAP3K7 Monoclonal Antibody

Western blot analysis of MAP3K7 using anti-MAP3K7 antibody (M01458).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human Hela whole cell lysates,
Lane 2: human K562 whole cell lysates,
Lane 3: human 293T whole cell lysates,
Lane 4: rat lung tissue lysates,
Lane 5: rat brain tissue lysates,
Lane 6: mouse lung tissue lysates,
Lane 7: mouse brain tissue lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-MAP3K7 antigen affinity purified monoclonal antibody (Catalog # M01458) at 1:500 overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for MAP3K7 at approximately 75 kDa. The expected band size for MAP3K7 is at 67 kDa.

calpain2a inhibits TRAF6 ubiquitin-ligase activity. a Ubiquitination of endogenous TRAF6 in MKC cells transduced with calpain2a-Flag and calpain2a-ΔCysPc-Flag and unchallenged (−) or challenged with LPS (+), assessed by immunoblot analysis with anti-ubiquitin after immunoprecipitation with anti-TRAF6 and input immunoblot analysis with indicated Abs. b , c HEK293 cells were cotransfected with TRAF6-HA and WT-ubiquitin-His or K63O-ubiquitin-His (in which only lysine 63 is kept) together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. d , e HEK293 cells were cotransfected with TRAF6-HA and WT-ubiquitin-His or K63O-ubiquitin-His (in which only lysine 63 is kept) together with calpain2a-Flag (0.5 μg, 1 μg), calpain2a-ΔCysPc-Flag (0.5 μg, 1 μg) or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. f Ubiquitination of overexpressed TRAF6 in MKC cells transduced with calpain2a-ΔCysPc-Flag, assessed by immunoblot analysis with anti-ubiquitin after immunoprecipitation with anti-Myc and input immunoblot analysis with indicated Abs. g , h HEK293 cells were cotransfected with TAK1-Myc, TRAF6-HA and WT-ubiquitin-His or K63O-ubiquitin-His (in which only lysine 63 is kept) together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. All the immunoprecipitates and input immunoblot analysis with anti-Myc, anti-Flag, anti-HA, and anti-Tubulin Abs. All experiments were performed in at least three independent experiments.
Index in PubMed under a CC BY license. PMID: 37002312

calpain2a inhibits TRAF6-mediated ubiquitination of ECSIT and BECN1. a HEK293 cells were transfected with ECSIT-Myc, TRAF6-Flag, or empty vector. After 24 h post-transfection, the cells were lysed and IP analyses with Flag antibody. b HEK293 cells were transfected with TAK1-Flag, ECSIT-Myc, or empty vector. After 24 h post-transfection, the cells were lysed and IP analyses with Myc antibody. c HEK293 cells were transfected with TRAF6-Myc, ECSIT-HA, empty vector, or different concentrations of calpain2a-ΔCysPc-Flag. After 24 h post-transfection, the cells were lysed and IP analyses with Myc antibody. d HEK293 cells were cotransfected with ECSIT-Myc, TRAF6-HA, WT-ubiquitin-His together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. e HEK293 cells were transfected with BECN1-Flag, TRAF6-Myc, or empty vector. After 24 h post-transfection, the cells were lysed and IP analyses with Myc antibody. f HEK293 cells were cotransfected with BECN1-HA, TRAF6-Myc, WT-ubiquitin-His together with calpain2a-Flag, calpain2a-ΔCysPc-Flag or empty vector. After 24 h post-transfection, the cells were lysed and purified with Ni-NTA agarose. The immunoprecipitates and input with anti-Myc, anti-Flag, anti-HA, and anti-Tubulin Abs. All experiments were performed in at least three independent experiments.
Index in PubMed under a CC BY license. PMID: 37002312

Immunofluorescent analysis of A431 cells, using TAK1 Antibody .