WB
Western blot analysis of Phospho-Ezrin (T567)/Radixin (T564)/Moesin (T558) on different lysates with Rabbit anti-Phospho-Ezrin (T567)/Radixin (T564)/Moesin (T558) antibody at 1/2000 dilution and competitor's antibody at 1/2000 dilution.
Lane 1: HeLa starved for 3 hours cell lysate,
Lane 2: HeLa starved for 3 hours then add 100nM Calyculin A for 30 minutes cell lysate,
Lane 3: NIH/3T3 starved for 24 hours cell lysate,
Lane 4: NIH/3T3 starved for 24 hours then add 100nM Calyculin A for 30 minutes cell lysate,
Lane 5: C6 cell lysate,
Lane 6: C6 treated with 100ng/mL Calyculin A for 1 hours cell lysate,
Lysates/proteins at 20 µg/Lane.
Exposure time: 9 seconds; 4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/2000 dilution and competitor's antibody at 1/2,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50000 dilution was used for 1 hour at room temperature.
ICC/IF
Immunocytochemistry analysis of HeLa cells untreated (upper, positive) and HeLa cells treated with 1μM staurosporine for 3 hours (lower, negative) labeling Phospho-Ezrin (T567)/Radixin (T564)/Moesin (T558) with Rabbit anti-Phospho-Ezrin (T567)/Radixin (T564)/Moesin (T558) antibody at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-Ezrin (T567)/Radixin (T564)/Moesin (T558) antibody at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (594) was used as the secondary antibody at 1/1000 dilution.
IHC
Immunohistochemical analysis of paraffin-embedded mouse liver tissue untreated / treated with λpp with Rabbit anti-Phospho-Ezrin (T567)/Radixin (T564)/Moesin (T558) antibody at 1/1000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody at 1/1000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
IHC
Immunohistochemical analysis of paraffin-embedded rat liver tissue untreated / treated with λpp with Rabbit anti-Phospho-Ezrin (T567)/Radixin (T564)/Moesin (T558) antibody at 1/1000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody at 1/1000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.