Cells were washed with cold PBS. Proteins were extracted with lysis buffer supplemented with a proteinase inhibitor. Cell lysates were centrifuged at 12,000 × g for 10 min at room temperature. Supernatants were collected and protein concentrations were quantified using a bicinchoninic acid kit (P0010S; Beyotime Institute of Technology). Then, proteins (35 mg) with loading buffer were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Western blots were developed using anti-ZIP4 (orb395779; Biorbyt, Cambridge, UK), anti-GAPDH (sc-32,233; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GJA1 (ab51067; Abcam, Cambridge, UK), anti-SGK1 (12,103; Cell Signaling Technology, Danvers, MA, USA), anti-MET (ab51067; Abcam), anti-VEGFC (ab83905; Abcam), mouse anti-rabbit IgG (7074; Cell Signaling Technology) and rabbit anti-mouse IgG (7076; Cell Signaling Technology).
Ab83905
Manufactured by Abcam
Sourced in United States
Ab83905 is a recombinant antibody product manufactured by Abcam. It is a mouse monoclonal antibody that can be used for research applications.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti mouse igg, by Cell Signaling Technology (2 mentions)
Anti gapdh sc 32233, by Santa Cruz Biotechnology (1 mentions)
Anti sgk1, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid kit, by Beyotime (1 mentions)
Ab32024, by Abcam (1 mentions)
Ab33766, by Abcam (1 mentions)
Ab13822, by Abcam (1 mentions)
Ab32527, by Abcam (1 mentions)
Donkey anti chicken igg irdye 680lt, by LI COR (1 mentions)
Irdye 800cw donkey anti rabbit igg, by LI COR (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Af457 sp, by R&D Systems (1 mentions)
Irdye 800cw donkey anti goat igg, by LI COR (1 mentions)
Ab199359, by Abcam (1 mentions)
Lentivirus, by Genechem (1 mentions)
4 protocols using ab83905
1
Protein Extraction and Western Blot Analysis
2
Immunohistochemical Staining of Paraffin-Embedded Tumor Samples
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Paraffin blocks that contained sufficient formalin-fixed tumor specimens were serial sectioned at 3 μm and mounted on silane-coated slides for immunohistochemical staining analysis. Dimethylbenzene rehydrated through 100% ethanol, 100% ethanol, 95% ethanol, 85% ethanol and 75% ethanol were applied to deparaffinize. In all, 0.01 mol/L of sodium citrate buffer (pH 6.0) was used to the progress of antigen retrieval treatment (autoclaved at 121°C, 2 min). Then, 3% H2O2 was applied to block endogenous peroxidase at room temperature for 10 min. The sections were washed in PBS solution subsequently and blocked with 10% goat serum (ZhongShan Biotechnology, Beijing, China) for 30 min and incubated with anti-eIF4E (ab33766, 1:100 dilution, monoclonal; Abcam, Cambridge, MA, USA) or anti-eIF4E-BP1 (ab32024, 1:100 dilution, monoclonal; Abcam) antibody or anti-vascular endothelial growth factor C (VEGFC; ab83905, 1:100 dilution, polyclonal; Abcam) or anti-phospho-4E-BP1 (Thr37/46) (236B4) (2855, 1:200 dilution, monoclonal; Cell Signaling Technology, Boston, MA, USA) at 4°C for 12 h. The sections were washed in PBS solution three times and incubated with HRP-conjugated secondary antibody for 30 min at room temperature. All slides were counterstained with diaminobenzidine (DAB) solution and 20% hematoxylin and dehydrated. The primary antibody diluent was regarded as negative control.
3
Protein Extraction and Western Blot Analysis
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Mice were anesthetized with ketamine/xylazine and transcardially perfused with PBS. The brain and spinal cords were quickly harvested. The brain and spinal cords were dounced using a tissue douncer with RIPA buffer (pH 7.5, 25 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) and the appropriate dilution of protease and phosphatase inhibitors. The dounced tissue was then sonicated and stored at −80 °C. Protein levels were normalized using a Bradford Assay and were assessed with Li-Cor Odyssey CLx infrared imaging system. Primary antibodies used for western blot were used as follows: goat anti-CCL21-unconjugated (1:100; AF457-SP; R&D Systems), rabbit anti-CCR7-unconjugated (1:100; ab32527; Abcam), rabbit anti-VEGFC-unconjugated (1:100; ab83905; Abcam), and chicken anti-ß-actin-unconjugated (1:1000; ab13822; Abcam). The appropriate secondary antibodies were used as follows: donkey anti-rabbit IgG-IRDye 800CW (1:2000; 926–32213; Li-Cor), donkey anti-goat IgG-IRDye 800CW (1:2000; 925–32214; Li-Cor), and donkey anti-chicken IgG-IRDye 680LT (1:2000; 926-68028; Li-Cor). For a detailed list of reagents, refer to Supplementary Table 1 . Representative uncropped western blots for VEGFC and ß-actin can be found in Supplementary Fig. 11 .
4
RAF1 Regulation of Lymphangiogenic Factors
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RAF1 was knocked down and overexpressed by transfection of FADU and SCC15 cell lines with lentivirus short hairpin RNA (shRNA) and overexpression vector. Lentiviruses were purchased from GeneChem (Shanghai, China), and the lentiviral composition was Ubi-firefly_Luciferase-IRES-Puromycin. The transfected cells were divided into four groups including sh-NC (knockdown negative control), sh-RAF1 (knockdown), vector (overexpression negative control) and lv-RAF1-OE (overexpression). FADU and SCC15 cells were seeded in 6-well plates and transfected using Lentiviruses on the following day when the cells were approximately 50–60% confluent. The multiplicity of infection (MOI) of the cells was 10. After 6 h transfection, the cell medium was cultured in DMEM supplemented with 10% FBS for 48 h. Then, the cells were cultured in medium containing 2 μg/mL puromycin to obtain stably infected cells. Next, the cells were harvested for qRT-PCR and WB (methods were the same as above) to detect the expression of RAF1. Primarry antibodies against lymphangiogenic cytokines VEGF-C (ab83905, Abcam, Cambridge, UK, diluted at 1:1000), LYVE-1 (ab219556, Abcam, Cambridge, UK, diluted at 1:1000) and PROX-1 (ab199359, Abcam, Cambridge, UK, diluted at 1:1000) were added to WB.
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