Rabbit anti ha tag

For immunostaining, HEK293T or HeLa cells grown on coverslips were fixed with 4% PFA in PBS at room temperature for 15 min and permeabilized with 0.25% PBST (PBS containing 0.25% Triton X–100) at room temperature for 15 min. Cells were blocked in 5% normal goat serum for 1 hr at room temperature. The samples were incubated overnight at 4°C with primary antibodies: rabbit anti-HA-tag (1:500; Cell Signaling Technology, #3724) and mouse anti-FLAG-tag (1:500; Sigma, F9291). Cells were washed in PBST and Alexa Fluor 488-anti-rabbit (1:200; Jackson Immunoresearch, 111545144) and Cy3-anti-mouse (1:200; Jackson Immunoresearch, 115165003) were used as the secondary antibodies. The samples were then stained with DAPI (1 μg/mL, Sigma, D9542) for 5 min at room temperature, washed, and mounted with Fluoromount aqueous mounting medium (Sigma, F4680).

For western blotting, cells were lysed with RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH8.0, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate) containing EDTA-free protease inhibitor cocktail (Roche). After centrifugation, clear supernatant was collected, added with protein sample buffer to final concentration of 50 mM Tris-HCl pH6.8, 2% SDS, 10% glycerol, 30 mM DTT, 0.002% bromophenol blue, boiled for 10 min and separated on SDS-PAGE gel. Proteins were semi-dry transferred onto PVDF membrane, blocked with 5% skim milk and probed with antibodies as indicated. For immunostaining, cells seeded on chamber-slides were fixed with 4% paraformaldehyde, 0.5% NP-40 permeabilized and blocked with 5% normal donkey serum (Jackson ImmunoResearch), followed by staining using the indicated antibodies. Images were captured using LSM880 confocal microscope (ZEISS). Mouse anti-FLAG-tag (Sigma-Aldrich), anti-beta-actin (Sigma-Aldrich), anti-GAPDH (Santa Cruz Biotechnology) and rabbit anti-HA-tag (Cell Signaling Technology), anti-IRF3 (Santa Cruz Biotechnology) antibodies were used.

Antibodies used for immunoblotting, IP, and IHC staining were as follows: Rabbit anti ZHX2 antibody (Genetex, 112232), Rabbit anti HIF1α (Cell Signaling, 36169), Rabbit anti HIF1β (Cell Signaling, 5537), Rabbit anti VHL (Cell Signaling, 68547), rabbit anti HA tag (Cell Signaling, 3724), mouse anti α-Tubulin (Cell Signaling, 3873), mouse anti Ubiquitin (Santa Cruz, sc-8017), and Rabbit anti NF-κB p65 (Cell Signaling, 8242S). Peroxidase conjugated goat anti-mouse secondary antibody (31430) and peroxidase conjugated goat anti-rabbit secondary antibody (31460) were from Thermo Scientific.

Cells were lysed with ice cold iCLIP Lysis Buffer^{3 (link)} containing 1X Protease Inhibitor Cocktail Set III (Milipore Sigma, Cat # 539134-1SET). Lysates were sonicated for 5 min at 30 second on/off intervals and protein was quantified using Pierce BCA Protein Assay Kit (Thermo Fisher, Cat # 23225). Total protein lysates were run on 4%–12% NuPAGE Bis-Tris gels in NuPAGE MOPS running buffer (Thermo Fisher, Cat # NP0050) and transferred to PVDF membranes. Membranes were blocked in 5% nonfat milk in TBST and incubated with the following primary antibodies: 1 h at room temperature with rabbit anti-HA-tag (Cell Signaling, Cat # 3724, Clone C29F4, Lot 10), washed 3X for 5 min with TBST, incubated for 2 h at RT in 5% nonfat dry milk powder in TBST with Rabbit TrueBlot: Anti-Rabbit IgG HRP (Rockland Immunochemicals, Cat # 18-8816-33, Clone eB182, Lot 46967), washed 3X for 5 min with TBST. Membranes were developed using the Azure C600 imaging system and Pierce ECL Western Blotting Substrate (Thermo Fisher, Cat # 32209).

After intracardial perfusion with 4% paraformaldehyde in PBS, pH 7.4, the brains were fixed overnight, placed in PBS with 30% sucrose for at least 24 h, and then sliced into 50 μm coronal or sagittal sections with a vibratome (Leica). The following primary antibodies were used: rabbit anti-HA tag (1:500; Cell Signaling), rat anti-D1R (1:500; Sigma-Aldrich), chicken anti-GFP/mVenus (1:1000; Abcam). The following secondary antibodies were used: goat anti-rabbit IgG-Alexa Flour 647 (1:750; Thermo Fisher Scientific), goat anti-rat IgG-Alexa 546 (1:750; Thermo Fisher Scientific), goat anti-chicken IgG-Alexa Flour 488 (1:1000; Thermo Fisher Scientific). Image acquisition was performed with a Zeiss LSM780 laser scanning confocal microscope using a 20× objective and on a Zeiss AxioImager M1 upright widefield fluorescence/differential interference contrast microscope with charge-coupled device camera using 5× objectives. Confocal images were collected using Zen Black 3.0 (Zeiss) and analyzed using Zen Blue 3.0 (Zeiss) and ImageJ. Sections were identified using landmarks and neuroanatomical nomenclature⁷⁴ . In some cases, AAV-injected mice received a dStr-infusion of 100 µM SNAP-Surface Alexa Fluor 647 (400 nL per hemisphere; NEB) followed by intracardial perfusion 24–48 h later.

Cells were washed three times with cold PBS and fixed in 4% paraformaldehyde for 15 minutes. Subsequently, cells were washed three times with PBS and incubated with 0.2% Triton X‐100 (Beyotime) for 20 minutes. After washing three times in PBS, cells were incubated with 1% BSA for 30 minutes at room temperature. The cells were then incubated overnight at 4ºC with the following antibodies: rabbit anti‐FGFR3 (1:50; Abcam Inc, Cambridge, MA, USA), rabbit anti‐HA tag (1:25; Cell Signaling, Beverly, MA, USA) and mouse anti‐MYC tag (1:25; Cell Signaling). Following this, cells were washed with PBST three times and were incubated with secondary antibodies (Donkey‐anti‐rabbit, Abcam Inc; Donkey‐anti‐mouse, Abcam Inc) for 1 hour at 37ºC. Finally, the cells were washed with PBST and observed under a Laser scanning confocal microscopy (Nikon A1R, Tokyo, Japan).