Cells were then washed in wash buffer, 5 mL formamide, and nuclease free water to a final volume of 50 mL for 5 mi nutes. Hybridization was reference 2 performed by mixing 100 uL of hybridization solution with a specific AhR probe and incubating the mixture overnight at 37 C. Cells were then stained with DAPI for 5 minutes. After washing, a drop of mounting solution was applied. The slides were then covered with the cell attached cover glasses and sealed with nail Inhibitors,Modulators,Libraries polish. Imaging was performed by confocal microscopy. RNA isolation and RT PCR Huh7 cells were seeded in six well Inhibitors,Modulators,Libraries plates, cul tured for 24 hours, and then incubated overnight in serum free medium. The cells were then treated with BBP for various times intervals. After stimula tion, cells were washed twice with PBS. Total RNA was extracted with TRIzol.
The RNA was applied to a Reverse Transcription System to synthesize Inhibitors,Modulators,Libraries cDNA. The cDNA was then amplified by specific primers. The PCR conditions were 95 C for 5 min, and then 34 cycles of 95 C for 30 sec, 54 C for 30 sec, and 72 C for 1 min, and a final extension at 72 C for 10 min. PCR products were separated on 2% agarose gels and visu alized using ethidium bromide. siRNA and shRNA transfection Immunoblot analysis Whole cell extracts were prepared in RIPA lysis buffer containing 1�� protease inhibitor cocktail. Protein concentrations were determined using a BCA protein assay kit. Equal amounts of protein were resolved by sodium dodecyl sulfate polyacaryamide gel electrophoresis, transferred onto a polyvi nylidene difluoride membrane, and blocked with 5% nonfat dry milk for 1 hour at room temperature.
After blocking, the membrane was incubated overnight with primary antibodies at 4 C and washed three times with PBST. The Inhibitors,Modulators,Libraries horseradish peroxidase conjugated second ary antibodies were incu bated for 1 hour at room temperature. The blots were washed three times with PBST and then visualized with an enhanced chemiluminescence kit. The primary antibodies were as fol lows AhR, Gq11, GB, PIP2, IP3R, and PI3K Inhibitors,Modulators,Libraries from Santa Cruz Biotechnology . p4442 MAPK, Akt, p Akt, NF��B, LaminAC, Histion H3 and tubulin from Cell Signaling Technology . COX 2 from Abcam . B actin from Sigma. Immunoprecipitation After preclearing for 30 minutes with protein G agarose, antibodies specific for Gq11, GB, or PI3K or IgG were added before overnight incubation at 4 C, followed by precipitation for 2 hours with protein G agarose.
The beads were washed three times with RIPA lysis buffer, boiled in sam ple buffer, and the protein were resolved by 8% SDS PAGE before performing immunoblot analysis of the indicated proteins. Transwell migration and invasion assays Cell migration assays were performed in 24 well in serts and cell invasion assays were performed KPT-330 Verdinexor (KPT-335)? in 24 well Matrigel Invasion inserts.