Rhodamine conjugated phallotoxin

Transfected cells were seeded onto coverslips in 12-well plates. Then, they were washed with PBS and fixed in 3.7% formaldehyde on ice for 10 min. Following this, cells were permeabilised in 0.5% Triton X-100 for 10 min, washed with PBS and incubated with 5 U/mL rhodamine-conjugated phallotoxin (Molecular Probes, Eugene, OR, USA) prepared in PBS (1:40 dilution) for 30 min before staining the nuclei with 1 μg/mL Hoechst 33342. F-actin cytoskeleton staining was evaluated and images were captured using a fluorescence microscope.

For western blot analysis, 48 hours after transfection, cells were harvested and lysed in lysis buffer [10 mmol/L Tris-HCl (pH 7.4), 1% SDS, 10% glycerol, 5 mmol/L MgCl2, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate, 5 μg/mL leupeptin, and 21 μg/mL aprotinin]. A total of 30 μg of protein lysates were separated by SDS-PAGE and transferred onto a PVDF membrane. The dilution of primary antibodies was according to the company's recommendation. Proteins were visualized using the enhanced chemiluminescence detection system.
Cells grown in cover glass were fixed with 4% paraformaldehyde and the nonspecific bindings were block by incubation with 1% bovine serum albumin. The glasses were probed with the first antibodies followed by TR- or fluorescein isothiocyanate-conjugated second antibodies. After mounting, the slips were visualized under an Olympus CKX 41 fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan).
For staining of F-actin, cells were washed with PBS and fixed in methanol/acetone (1:1) for 5 min on ice, and incubated with rhodamine-conjugated phallotoxin (5 U/mL, Molecular Probes) in PBS at a 1:40 dilution for 1 h. Coverslips were washed, mounted, and visualized using fluorescence microscope. Nuclei were stained with 1 μg/mL Hoechst 33258 and cells were analyzed using fluorescence microscope.

For western blot analysis, cells were harvested and lysed in lysis buffer. A total of 30 μg of protein lysates was separated by SDS-PAGE and transferred onto a PVDF membrane. Primary antibodies were diluted according to the company’s recommendation. Protein bands were detected using ECL Western Blotting Detection Reagent (GE Healthcare).
Cells grown on cover glass were fixed with 4% paraformaldehyde, and nonspecific binding was blocked by incubation with 1% bovine serum albumin. The glasses were probed with the primary antibodies prior to incubation with TR- or fluorescein isothiocyanate-conjugated second antibodies. After mounting, the slips were visualized under an Olympus CKX 41 fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan) or a Zeiss LSM710 confocal microscope using the 40x or 63x objectives (ZEISS International, Germany).
For staining of F-actin, cells were washed with PBS, fixed in methanol/acetone (1:1) for 5 min on ice, and incubated with rhodamine-conjugated phallotoxin (5 U/mL, Molecular Probes) in PBS at a 1:40 dilution for 1 h. Coverslips were washed, mounted, and visualized using fluorescence microscope. Nuclei were stained with 1 μg/mL Hoechst 33258, and cells were analyzed using a fluorescence microscope.