Rabbit anti vimentin

For Co-IP assays, HEK-293T cells were transfected as indicated: 36 h later, cells were lysed in IP buffer (1% Triton X-100, 150 mM NaCl, and 50 mM Tris/HCl (pH 7.5), 1 mM PMSF, and protease inhibitor cocktail) and incubated with anti-Flag M2 affinity gels for 4 h at 4 °C. For reverse IP, HEK-293T cells were lysed and incubated with anti-HA antibody and protein A/G agarose for 12 h at 4 °C. Agarose was washed five times with IP assay buffer, boiled in SDS sample buffer, and subjected to Western blot analysis. Western blotting and immunofluorescence (IF) were performed as previously [40 (link)].
The following antibodies were used: mouse anti-Flag (F3165, Sigma-Aldrich), rabbit anti-Flag (F7425, Sigma-Aldrich), rabbit anti-GST (2622, Cell Signaling Technology), mouse anti-β-actin (66009-1-Ig, Proteintech), rabbit anti-Snail (3879, Cell Signaling Technology), mouse anti-Snail (sc-271977, Santa Cruz), rabbit anti-p66β (ab76925, Abcam), anti-biotin, HRP-linked (7075, Cell Signaling Technology), and rabbit anti-IgG (2729, Cell Signaling Technology), mouse anti-α-tubulin (66031-1-Ig, Proteintech), rabbit anti-Fibronectin1 (15613-1-AP, Proteintech), rabbit anti-E-cadherin (20874-1-AP, Proteintech), rabbit anti-Vimentin (10366-1-AP, Proteintech).

Cells seeded on coverslips were fixed in 4% paraformaldehyde in DPBS at RT followed by permeabilization with 0.5% of Triton X-100 (Santa Cruz Biotechnology, Dallas, TX, USA) in DPBS 1x (PBTx) at RT for 5 min. Fixed cells were then blocked with 1% Bovine Serum Albumin (BSA) (Sigma-Aldrich) and 5% Normal Donkey Serum (NDS) (Sigma-Aldrich) in PBTx for 30 min at RT in darkness and incubated with primary antibodies diluted in blocking solution overnight at 4 °C in darkness. After primary antibody washing, secondary antibodies diluted in blocking solution were incubated for an hour at RT in darkness. Cells were stained and mounted on microscope slides using Fluoroshield with DAPI (Sigma-Aldrich). Immunostaining was performed using the following primary antibodies: rabbit anti-vimentin (1:500; Proteintech, Manchester, UK), mouse anti-alpha smooth muscle actin (α-SMA) (1:1000; Abcam) and mouse anti-myosin 4 (1:500; Invitrogen). Secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 594 (ThermoFisher Scientific, Waltham, MA, USA). Immunofluorescence analysis was performed using Olympus BX51 Fluorescence Microscope (Olympus) and cell images were acquired by DPController and DPManager software (Olympus).

Cells were washed with 1× PBS for 3 minutes, fixed with 4% paraformaldehyde for 20 minutes and washed for three times with 1× PBS for 3 minutes. Then, cells were treated with 0.5% Triton X‐100 for 20 minutes at room temperature (this step was omitted when the antigen is expressed on the cell membrane) and washed with 1× PBS. Blocking was performed by normal goat serum for 30 minutes, followed by addition of primary antibody: rabbit anti‐E‐cadherin (1:400 dilutions, ProteinTech, China), anti‐N‐cadherin (1:300 dilutions, ProteinTech), rabbit anti‐Vimentin (1:200 dilutions, ProteinTech), rabbit anti‐VE‐cadherin (1:200 dilutions, ProteinTech) and rabbit anti‐VEGFA (1:200 dilutions, ProteinTech) to incubate overnight at 4°C, and then washed for three times with PBS. Add 1:200 fluorescent secondary antibody: goat anti‐Rabbit Alexa 488 (1:200 dilutions, ZSGB‐BIO, China), goat anti‐Rabbit Alexa 594 (1:200 dilutions, ZSGB‐BIO) and incubated at 37°C in dark for 2 hours. Add DAPI, incubated in dark for 5 minutes, washed with PBS for three times, and observed under a fluorescence microscope.

The renal tissue and cells are lysed on ice with lysis buffer, and protein concentration was determined. The samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to PVDF membrane (Microporous, USA). Subsequently, the membrane was blocked for 2 h, and probed with primary antibody at 4°C, such as rabbit-anti-fibronectin (1:1,000), rabbit-anti-Shh (1:1,000; Proteintech), rabbit-anti-Shh (1:500; Santa Cruz), rabbit-anti-TNF-α (1:1,000; Santa Cruz), rabbit-anti-MCP-1 (1:1,000, Proteintech), rabbit-anti-PCNA (1:1,000; Proteintech), rabbit-anti-Vimentin (1:1,000; Proteintech), mouse-anti-p21 (1:1,000; Santa Cruz), mouse-anti-p16^INK4A (1:1,000; Santa Cruz), rabbit-anti-PDGFR-β (1:1,000; Santa Cruz), rabbit-anti-β-actin (1:8,000; Proteintech), and anti-GAPDH antibody (1:5,000; Wuhampmack Biotechnology Co., Ltd., China) for 16 h at 4°C. The chemiluminescence signal was detected after incubation with second antibody for 1 h. Finally, ImageJ software were used for analysis.

Equivalent extracted amounts of protein were separated with polyacrylamide SDS gels (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific). They were then probed with different primary antibodies, including rabbit anti-CHN1 (1:150, Abcam), mouse anti-fibronectin (1:350, Invitrogen), rabbit anti-β-catenin (1:1000, CST, USA), rabbit anti-Snail (1:500, CST), rabbit anti-vimentin (1:1000, Proteintech Group), rabbit anti-E-cadherin (1:5000, Proteintech Group), rabbit anti-p-Akt (1:2000, CST), rabbit anti-Akt (1:1000, CST), and rabbit anti-p-GSK-3β (1:1000, CST), and the blots were detected with peroxidase-conjugated secondary antibodies (ZSGB-Bio). Signals were acquired using a chemiluminescence kit (Merck Millipore, Germany). Rabbit anti-β-tubulin (1:10,000) and mouse-anti GAPDH (1:10,000, both from Transgen Biotech, China) were used as internal controls. Universal anti-mouse/rabbit secondary antibodies were purchased from ZSGB-Bio (1:4000). To inhibit the PI3K/Akt pathway, the cells were treated with 5 μM PI3K inhibitor LY294002 (MedChem Express, USA) for 12 h.

Total protein was extracted from three infected CRC cell lines (HCT116, RKO, and MC38) using RIPA lysis buffer supplemented with 1% PMSF. Total protein was separated using a 10% sodium dodecyl sulfate‒polyacrylamide gel and then transferred to a PVDF membrane. The membranes were blocked with 5% skim milk for 2 h and then incubated with rabbit anti-C6orf15 (Proteintech, USA; 1:1000 dilution), rabbit anti-β-catenin (Proteintech, USA; 1:1000 dilution), rabbit anti-ZEB1 (Abmart, Shanghai; 1:1000 dilution), rabbit anti-E-cadherin (Abmart, Shanghai; 1:1000 dilution), rabbit anti-N-cadherin (Abmart, Shanghai; 1:1000 dilution), rabbit anti-Vimentin (Proteintech, USA; 1:1000 dilution), rabbit anti-ZO-1 (Abmart, Shanghai; 1:1000 dilution), mouse anti-GAPDH (Abmart, Shanghai; 1:3000 dilution), rabbit anti-CPT1A (Proteintech, USA; 1:1000 dilution), and rabbit anti-LaminB1 (Proteintech, USA; 1:1000 dilution). The membranes were then incubated with secondary antibodies for 2 h. We assessed the protein blotting results using an enhanced chemiluminescence (ECL) assay kit (Thermo Fisher Scientific, Rockford, IL, USA), and each experiment was performed three times.

Western blot was performed as described in previous study.¹⁴ Afterwards, the membrane was incubated with the enzyme‐labelled secondary antibody at room temperature for 1 hour, and the ECL kit (Thermo Scientific, Rockford, IL, USA) was used for detection. Antibodies used: rabbit anti‐CUL4B (Proteintech, 12,916‐1‐AP), rabbit anti‐E‐Cadherin (Proteintech, 20,874‐1‐AP), rabbit anti‐Vimentin (Proteintech, 10,366‐1‐AP), and mouse anti‐GAPDH (Proteintech, 60,004‐1‐Ig). For proteins with significantly different molecular weights, PVDF membranes are tailored to incubate different antibodies.