Hs101 01

Protein extracts were denatured at 95 °C for 5 min and separated on 10% SDS–PAGE gels at 110 V for approximately 70 min. The proteins were transferred to nitrocellulose (NC, HATF00010, Millipore) filters at 80 V for 5 h. The NC membrane was initially blocked with 5% nonfat milk and 2% goat serum (16210064, Thermo Fisher(v/v)) in tris-buffered saline with 0.1% Tween 20 (93773, Sigma) at room temperature for 1 h. Monoclonal antibodies to β-actin (HC201-02, TransGen Biotech), polyclonal antibody to Syt1 (CSB-PA019553GA01HU, CUSABIO Biotech), rabbit polyclonal anti-phosphotyrosine (Cat# PTM-702, PTM BIO) were employed for Western blot analyses as primary antibodies at 4 °C overnight. After three washes of 5 min each with tris-buffered saline with 0.1% Tween 20, either goat anti-rabbit (HS101-01, TransGen Biotech) or anti-mouse (HS201-01, TransGen Biotech) immunoglobulin G was added at a dilution of 1:5000 as the secondary antibody. The NC membrane was scanned with an imaging system (MicroChemi 4.2, DNR Bio Imaging Systems).

Cell extracts from the adhered mycelium mat incubated for 18 h were used for performing co-immunoprecipitation analyses. Protein extraction, quantification, and co-immunoprecipitation assays were performed as described previously (47 (link)). Briefly, the 4 mg/ml protein extracts in extraction buffer were incubated with 5 μl of monoclonal antibody to c-Myc (HT101-02, TransGen Biotech), 10 μl of antibody to NC2α, 10 μl of antibody to NC2β, 10 μl of antibody to INO80, 10 μl of antibody to ARP8, 10 μl of antibody to IgG (HS101–01, TransGen Biotech) for 4 h at 4°C with rotation. Then the 40 μl precleaned protein G-Sepharose (17-0885-02, GE Healthcare) were added and incubated for 1 h at 4°C with rotation. The beads were washed three times with ice-cold extraction buffer, mixed with protein loading buffer, and boiled for 10 min, and the immunoprecipitated proteins were analyzed by Western blotting.

Western blot analysis was performed as previously described.⁵⁹ (link) The primary antibodies used were anti-GFP (50430-2-AP, 1:1,000, Proteintech), anti-FLAG (AF519, 1:1,000, Beyotime), anti-MYOD (ABP53067, 1:500, Abbkine), anti-MyHC (B103, 0.5 μg/mL, DHSB), anti-SMURF2 (ab94483, 1 μg/mL, Abcam), anti-SMAD2/3 (70R-51804, 1:500, Fitzgerald), anti-phosphorylated (phospho-)SMAD2 (bs-3419R, 1:500, Bioss), anti-phospho-SMAD3 (bs-3425R, 1:500, Bioss), anti-β-actin (bsm-33036M, 1:1,000, Bioss), and anti-GAPDH (60004-1-Ig, 1:5,000, Proteintech). ProteinFind goat anti-mouse immunoglobulin G (IgG) (H+L), horseradish peroxidase (HRP) conjugate (HS201-01, 1:1,000, TransGen Biotech, Beijing, China) and ProteinFind goat anti-rabbit IgG (H+L), HRP conjugate (HS101-01, 1:500, TransGen Biotech) were used as a secondary antibody.
The immunofluorescence was performed using anti-FLAG (AF519, 1:1,000, Beyotime) or anti-MyHC (B103, 2.5 μg/mL, DHSB). Images were obtained with a fluorescence microscope (DMi8; Leica, Germany). The area of cells labeled with anti-MyHC was measured and calculated as previously described.⁵⁹ (link)

Protein extracts were separated by 12% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (PALL) by semi-dry blotting in 15 mM Tris–HCl, 120 mM glycine, and 20% methanol. The membrane was blocked with non-fat dry milk in Tris-buffered saline (0.1% Tween-20). Blots were probed with specific primary antibodies and horseradish-peroxidase-conjugated secondary antibodies against mouse immunoglobulin G (TransGen Biotech HS201-01, 1:5000 dilution) and rabbit immunoglobulin G (TransGen Biotech HS101-01, 1:5000 dilution). Chemiluminescence detection was performed with an ECL kit (Amersham, Piscataway, NJ, USA) following the manufacturer’s instructions.

The sections were heated at 70°C for 1 h, dewaxed in xylene, and dehydrated through a gradient concentration of alcohol. After retrieving and blocking endogenous peroxidase and non-specific staining with 3% H₂O₂ and normal bovine serum, the sections were incubated with primary antibody overnight at 4°C. The slides were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (HS101-01, TransGen Biotech, 1:500) for 10 min at 37°C. Finally, the sections were visualized by diaminobenzidine (DAB) solution for 15 min at 37°C and then counterstained with hematoxylin. Two pathologists blinded to the patient outcomes independently scored the staining intensities and percentages of positive tumor cells. The results of immunohistochemistry were observed using an optical microscope (ZTX-3S-C2, AS ONE Corporation). Specimens were stained with antibodies to QPCT (ab201172, Abcam, 1:100), PIK3CA (ab135384, Abcam, 1:100), CD31 (ab28364, Abcam, 1:50) and CD34 (ab110643, Abcam, 1:100).

The aorta arch cells or endothelial cells of mice were mixed with the lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) to isolate total protein. The proteins were then dissolved in 2× sodium dodecyl sulfate (SDS) loading buffer, separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane. The membrane was incubated with primary antibodies rabbit anti-proliferating cell nuclear antigen (PCNA) (ab92552, 1:1,000), cleaved-caspase 3 (ab2302, 1:1,000), Bcl-2-associated X protein (Bax) (ab182733, 1:2,000), B-cell lymphoma 2 (Bcl-2) (ab692, 1:500), HIF-1α (ab2185, 1:1,000), and β-actin (ab8226, 1:1,000) (all from Abcam, Cambridge, UK). Next, the membrane was washed with tris-buffered saline Tween-20 (TBST) and incubated with the horseradish peroxidase-labeled secondary rabbit anti-antibodies (HS201-01, Mouse; HS101-01; TransGen Biotech, Beijing, China) for 1 h. Then, the membrane was rinsed with TBST, developed using an enhanced chemiluminescence fluorescence detection kit (BB-3501, Amersham Pharmacia Biotech, Inc., Cambridge, UK) and photographed using a Bio-Rad Image Analysis System (Bio-Rad Laboratories, Hercules, CA, USA). The relative protein expression was calculated using the Quantity One v4.6.2 software.