Ha tag c29f4 rabbit mab

For yeast experiments, 1–2 ml of yeast cells in the indicated background and medium was collected by centrifugation and snap frozen in liquid nitrogen for storage. Pellets were disrupted, boiled in 120 μl 1X LDS sample buffer (Thermo) for 10 min, and vortexed with an equal volume of 0.5 mm acid-washed glass beads to break cells at 4 °C for 2 min with a 1 min interval. Cell lysates were boiled for 5 min, separated from glass beads by 15,000 g centrifugation at room temperature for 30 sec, and analyzed by SDS-PAGE. For mammalian data, RPE-1 cells were washed with PBS and lysed with RIPA buffer (MilliporeSigma) supplemented with protease inhibitors on ice for 20–30 min. Cell lysates were further sonicated and incubated on ice for 5 min, followed by 10 min 21,200 g centrifugation at 4 °C. The supernatant was collected and analyzed by SDS-PAGE.
Transfer was performed using iBlot2 (Thermo) and immunoblots were developed using Clarity Western ECL substrate (Bio-Rad) for HRP-linked secondary antibodies, or directly using fluorescent IRDye secondary antibodies (LI-COR). Images were acquired by using LI-COR imaging systems and analyzed in Image Studio (LI-COR). HA-tag (C29F4) rabbit mAb #3724 from Cell Signaling Technology. PGK1 mouse mAb (22C5D8) from Invitrogen. FLAG mouse clone M2 (F1804) from MilliporeSigma. GFP Living Colors A.v. mAb clone JL-8 (632381) from Takara Bio.

Cells transformed with the Rpb1-Flag-HA construct were fixed in 4% paraformaldehyde in 1× PHEM at room temperature for 25 min. Then, cells were washed thrice with 1× PBS and permeabilized with 1% Triton X-100 in 1× PHEM for 1 h. After permeabilization, the cells were blocked in blocking solution (3% bovine serum albumin (BSA) and 1% Triton X-100 in TBSTEM) for 1 h and incubated with the primary antibody (HA-Tag (C29F4) Rabbit mAb, Cell Signaling Technology) at a dilution of 1:400 in blocking solution overnight at 4 °C. After three washes with blocking solution for 15 min, the cells were incubated with the secondary antibody (Alexa Fluor 546 Goat anti-rabbit IgG, Invitrogen) at a 1:4000 dilution in blocking solution for 45 min at room temperature. Lastly, the cells were washed with 1× PBS and stained with DAPI. Microscopy images were captured with an Axio Imager D2 microscope (Zeiss, Germany).

Whole adults were homogenized in ice-cold RIPA buffer (P0013B, Beyotime) supplemented with 100 μM protease inhibitors PMSF (ST506, Beyotime). Total protein concentrations were measured using the BCA assay Kit (P0012S, Beyotime). Protein samples were diluted to equal concentrations using lysis buffer and loaded into SDS-PAGE gels for Western blot analysis. The primary antibodies were HA-Tag (C29F4) rabbit mAb (1:8000, #3724, Cell Signaling) and mouse monoclonal anti-α-tubulin (1:50,000, Sigma, T6074). The secondary antibodies were goat anti-rabbit IgG H&L (HRP) (1:20,000, ab205718, Abcam, Cambridge, UK) and goat anti-mouse IgG (H + L), HRP (1:20,000, BE0102, Easybio, Beijing, China).

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.

To investigate the interaction between Cspg4 and Wnt11, HEK-293T cells were transfected with pT7-cspg4^stop-FLAG-IRES-dsRed or pT7- wnt11^stop-HA-IRES-dsRed, or co-transfected with both plasmids as above described. After total protein was extracted by lysis buffer, Anti-FLAG M2 Magnetic Beads (Sigma-Aldrich) were used to precipitate flag-tagged Cspg4 protein according to the manufacturer’s instruction. After immunoprecipitation, samples were separated by 12% SDS-PAGE or 3~8% tris-acetate gel for detecting HA-tagged Wnt11 or flag-tagged Cspg4, respectively. Protein was transfer onto PVDF membranes and blocking with 5% bovine serum albumin (Sigma-Aldrich). After blocking, membrane was hybridized with HA-Tag (C29F4) Rabbit mAb (#3724, Cell Signaling Technology, Danvers, MA, USA) or DDDDK tag antibody (ab21536, Abcam), and a 1:5000 dilution of goat anti-rabbit antibody (PerkinElmer Inc.) was used as secondary antibody. The signal was detected by Chemiluminescence Reagent.