Alexa fluor 594 affinipure goat anti rabbit igg h l

The cells were washed three times with PBS and were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 20 min, blocked in PBST containing 5% BSA for 1 h at 37°C, and then incubated with primary antibody at 37°C for 1 h. Then, the cells were incubated with a second antibody at 37°C for 1 h. Actin filaments were stained with TRITC-phalloidin (2 μg/mL) for 30 min at 37°C. Cell nuclei were stained with DAPI (1 μg/mL) for 10 min. The cells were visualized using a confocal laser scanning microscope (LEICA TCS SP8, Germany).
HA-Tag (C29F4) rabbit MAb (Cell signaling technology. catalog number 3724) was used at a 1:200 dilution. DYKDDDDK Tag (9A3) mouse MAb (Cell Signaling Technology. catalog number 8146) was used at a 1:200 dilution. MYC-Tag antibody (Proteintech. catalog number 16286-1-AP) was used at a 1:200 dilution. NDV NP monoclonal antibody (gifted by Chan Ding) was used at a 1:100 dilution. Beta catenin polyclonal antibody (Proteintech. catalog number 51067-2-AP) was used at a 1:100 dilution. Alexa Fluor 594 AffiniPure Goat Anti-Rabbit IgG (H+L) (Yeasen catalog number 33112ES60) was used at a 1:200 dilution. Alexa Fluor 594 AffiniPure Goat Anti-Mouse IgG (H+L) (Yeasen catalog number 33212ES60) was used at a 1:200 dilution. Alexa Fluor 488 AffiniPure Goat Anti-Mouse IgG (H+L) (Yeasen catalog number 33206ES60) was used at a 1:200 dilution.

Gallic acid (GA, 98%), ellagic acid (EA, 98%) and catechinic acid (CA, 98%) were from Chengdu PUSH Bio-technology Co., Ltd. (Chengdu, Sichuan, China). Ziyuglycoside I (98%) and ziyuglycoside II (98%) were purchased from Chengdu PureChem-Standard Co., Ltd (Chengdu, Sichuan, China). DEPC solution obtained from Sangon Biotech (Shanghai, China). Wnt3a protein was purchased from EMPOWERING STEM CELL R&D (Burlingame, CA, USA). The CCK-8 kit was obtained from Dojindo (Mashikimachi, Kamimashiki Gun Kumamoto, Japan) and Steady-Glo® Luciferase Assay System was from Promega Corporation (Madison, WI, USA). PrimeSCript™ RT reagent Kit with gDNA Eraser and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) were purchased from Takara Bio Inc (Kusatsu, Shiga, Japan). RIPA reagent, Anti-β-catenin (#19807) and anti-β-actin (#4970) were from Cell Signaling Technology, Inc. (Danvers, MA, USA). Alexa Fluor 594 AFFINIpure Goat Anti-Rabbit IgG (H + L) and DAPI Fluoromount-Gtm were obtained from YEASEN Co., Ltd (Shanghai, China).

The cells were lysed with lysis buffer (Beyotime, Shanghai China) supplemented with a protease inhibitor solution (Beyotime) and centrifuged at 12 000 g for 10 min at 4 °C. Extracted proteins were separated on 10–15% sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes. After blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at 37 °C, the membranes were incubated overnight with primary antibodies at 4 °C, washed with TBST, and then incubated with secondary antibodies. The western blot results were quantified by densitometric analysis using the Quantity One 4.6.9 software (Bio-Rad).
The following antibodies were used: anti-cleaved caspase-9 (9509T, CST), anti-cleaved caspase-3 (9664T, CST), anti-Bax (#2772, CST), anti-Bcl2 (#3498, CST), anti-cytochrome c (10093, Proteintech), VDAC rabbit mAb (4661, CST), anti-MFN1 (NBP1-71775, Novus Biologicals), DRP1 rabbit mAb (8570, CST), OPA1 rabbit mAb (80471, CST), anti-FIS1 (D122377-0025, BBI life sciences), anti-MFN2 (12186, Proteintech), anti-beta tubulin (10094, Proteintech), anti-PSD95 (20665, Proteintech), spinophilin rabbit mAb (14136, CST, Boston), HP-goat anti-mouse (ZB-2305, Zsbio, Beijing, China), HP-goat anti-rabbit (ZB-2301, Zsbio, Beijing, China), and Alexa Fluor 594 AffiniPure Goat Anti-Rabbit IgG (H+L) (33112ES60, Yeasen).