The cellular expression of the six candidate proteins were verified experimentally via Western Blot analysis. Total cell lysates were obtained using RIPA buffer (Thermo Fisher Scientific, United States). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, United States). The membranes were blocked in PBS, 10% (w/v) skim milk for 1 h in phosphate buffer saline-Tween 20 (PBS-T), and incubated for 3 h at RT in 5% milk in PBS-T with primary antibodies: CDH2, EGFR, ITGA3, ITGA5, ITGB1, and CALR (Abcam, Cambridge, United Kingdom). Then, after washing, the PVDF membrane was incubated with secondary antibody (The Jackson Laboratory, United States) for 40 min at RT. Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific, United States) was used for visualization.
Itgb1
Manufactured by Abcam
Sourced in United States
ITGB1 is an integrin beta chain protein that is a component of the integrin cell surface receptors. Integrins mediate cell-cell and cell-matrix adhesion and play a role in a variety of cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Itga5, by Abcam (2 mentions)
Ywhaz, by Absin (2 mentions)
Itga2, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Itga3, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence advanced system, by GE Healthcare (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Tgf β, by Abcam (1 mentions)
Bca protein concentration measurement kit, by Thermo Fisher Scientific (1 mentions)
Ctla4, by Abcam (1 mentions)
Pdcd 1, by Abcam (1 mentions)
Tigit, by Abcam (1 mentions)
Itgal, by Abcam (1 mentions)
Il 10, by Abcam (1 mentions)
Histone 3, by Abcam (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Tsg101, by Abcam (1 mentions)
Calnexin, by Abcam (1 mentions)
10 protocols using itgb1
1
Protein Expression Verification by Western Blot
2
Protein Expression Analysis Protocol
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Protein lysates of Vector‐TEVs, Foe‐TEVs or Foe‐Th cells were prepared according to standard procedures, and the contents of proteins were assessed by the BCA protein concentration measurement kit (Thermo Fisher Scientific). Subsequently, the samples were isolated using Bis‐Tris gel (Invitrogen), and then transferred to polyvinylidene difluoride membranes (Millipore). The membrane was blocked in TBS‐T in 5% BSA for 1 h, incubated with primary antibodies overnight at 4 °C, then rinsing the membranes with PBS. Then, the membranes were covered with secondary antibody. Incubated at room temperature for 2 h, signals were assessed by enhanced Chemiluminescence Advanced System (GE Healthcare). The primary antibodies involving against CD9 (1:1000, Abcam), CD63 (1:1000, Abcam), Tubulin (1:1000, Abcam), TSG101(1:1000, Abcam), CTLA‐4 (1:1000, Abcam), TGF‐β (1:1000, Abcam), IL‐10 (1:1000, Abcam), PDCD‐1 (1:1000, Abcam), CD35 (1:1000, Abcam), CD73 (1:1000, Abcam), LAG3 (1:1000, Abcam), TIGIT (1:1000, Abcam), calnexin (1:1000, Abcam), histone 3 (1:1000, Abcam), and GM13 (1:1000, Abcam), ITGA4 (1:800, Abcam), ITGAL (1:800, Abcam), ITGB1 (1:800, Abcam), ITGB2 (1:800, Abcam) and secondary antibody Alexa Fluor Plus 800 (1:10 000, Thermos) were used.
3
Immunohistochemistry of Tumor Biomarkers
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Sections of formalin-fixed, paraffin-embedded tumors were used. Briefly, after antigen retrieval, the following primary antibodies were used: ITGAV (sc-376,156, Santa Cruz,); ITGB1 (AB3167, Abcam), ITGB6 (HPA023626, Atlas), CEACAM7 (LS-B13068, LSBio), TGFBR1 (PA5–98192, Thermo Scientific), phospho-SMAD2 (AB3849, Millipore), TGFBI (MA526731, Thermo Fisher Scientific), Twist (ab50581, Abcam), Ki 67 (M7240, Agilent); human-specific fibronectin (NCL-FIB, Leica Biosystems); fibronectin, cross-reacting with the fibronectin-equivalent protein in mouse (A0245, Dako); STAT1 (HPA000931, Sigma-Aldrich), HLA-DR (M0746, Agilent). Vectastain ABC kit (Vector) was used. Slides were scanned by Axio Scan Z1 (Zeiss). For quantification with Image J (NIH), images of four randomly chosen vision fields (not containing necrotic areas) were analyzed. If artifacts interfered with automated quantification by Image J, the staining was quantified by two experienced investigators: grade 0: no reaction or weak focal reaction; grade 1 intense focal or diffuse weak reaction; grade 2 moderate diffuse reaction; grade 3 for an intense diffuse reaction.
4
Immunohistochemical Analysis of LIHC Tissues
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Tumor tissues and matched non-tumor tissues derived from 10 LIHC patients were incubated in 4% paraformaldehyde overnight, followed by embedding in paraffin. The tissues were sectioned at a thickness of 4 μm. Then, the tissues were processed by deparaffinization, rehydration, and antigen retrieval. The sections were blocked in a commercial goat serum (Boster Biological Technology, United States) and incubated with the following antibodies: ITGB1 (1:1,000; Abcam, United States), PXN (1:1,000; Abcam, United States), and YWHAZ (1:1,000; Absin, China) at 4°C overnight. The next day, we used SP Rabbit and Mouse HRP Kit (CWBIO, China) to amplify signal. The sections were then counter-stained with hematoxylin, dehydrated with a graded ethanol series, and cleared with xylene. Images were acquired by Aperio CS2 Digital Pathology Slide Scanners (Leica, Germany). The analysis of IHC staining signal was performed by ImageJ.
5
Comprehensive Tissue Analysis Protocol
6
Immunohistochemical Analysis of Cell Adhesion Markers
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Tumor tissue sections were deparaffinized and heat-treated with citrate buffer, pH 6.0, for 5 min as an epitope retrieval protocol. The tissue sections were then exposed to 0.03% hydrogen peroxide for 5 min to block endogenous peroxidase activity followed by incubation with 2% BSA to block nonspecific antibody binding sites. Then the tissue sections were incubated with ITGA1, ITGB1, VCL and ILK antibodies (Abcam, Cambridge, MA, USA) at proper concentration according to manufacturer's instructions for 2 h at 37°C. HRP-conjugated anti-mouse/rabbit antibody (Multi Science, Hangzhou, China) was then added for 1 h and the color was developed using 3-30-diaminobenzidine. Following washing step, the sections were counterstained with hematoxylin, washed and dipped briefly in a water bath containing drops of ammonia, prior to dehydration and mounting in Diatex. The stained sections were analyzed and scored using a Leica Microscope (Leica, Wetzlar, Germany). Scoring was based on the intensity and average percentage of positive cells. The staining intensity was scored with “1” (negative or weakly positive), “2” (moderately positive) and “3” (strongly positive). The average percentage of positive cells was scored as: 1 (<25%), 2 (25-50%), 3 (50-75%) and 4 (>75%).
7
Integrin Beta 1 Knockdown in HCC Cells
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HCC cells were collected after being transfected with ITGB1-specific siRNA or negative control siRNA for 72 h. Then, these cells were lysed in the RIPA buffer (Solarbio, Beijing, China). For each sample, 70 μg protein was separated by SDS-PAGE, followed by transferring to PVDF membranes. Then, the membranes were incubated with the following antibodies: ITGB1 (1:1,000; Abcam, United States), PXN (1:1,000; Abcam, United States), YWHAZ (1:1,000; Absin, China), and AKT (1:1,000; Cell Signaling Technology, United States) at 4°C overnight. The next day, we incubated the membranes with HRP-conjugated secondary antibody (1:1,000; Cell Signaling Technology, United States) at room temperature for 1 h. The ECL detection kit (Affinity Biosciences, China) was employed to examine the specific proteins. We used GAPDH (1:1,000; Cell Signaling Technology, United States) as loading controls.
8
Immunofluorescence Imaging of Rat Liver Samples
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Cryosections of young, old, regenerating (PHx), and perfused rat livers as well as isolated HSC under static and shear stress conditions were fixed in ice‐cold methanol or 4% formalin (n = 3). The following primary antibodies were used: COL1 (C2456; Sigma‐Aldrich), COL4 (ab6586; Abcam), LAMA5 (NBP2‐42391; Novus Biologicals), FN (610078; Becton Dickinson), ITGA5 (ab150361; Abcam), ITGB1 (ab52971; Abcam), HGF (80429‐R052; Sino Biological), GFAP (MAB3402; Merck/Millipore), desmin (ab8592; Abcam), and α‐SMA (M0851; Dako/Agilent). The samples were then incubated with anti‐mouse or anti‐rabbit secondary antibodies labeled with Cy3 or FITC (Millipore). The tissue sections were mounted with Fluoromount G containing 4′,6‐diamidino‐2‐phenylindole (DAPI; Southern Biotech) and a glass coverslip. Images were taken using an Olympus IX50 microscope equipped with a DP71 camera (Olympus). The same exposure time was used for each antibody to enable comparison of liver sections from different age groups.
9
Jaceosidin Inhibits Lung Cancer Progression in Mice
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Male BALB/c nude mice (aging from 4 to 6 weeks, 18–22 g) were provided by the HFK Biotechnology company. All animal experimentation was approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University. The A549 cell suspension (1 × 106 cells/200 μL/mouse) containing PBS (Gibco, Invitrogen, USA) and Matrigel was fully re-suspended and injected into the axilla of the mice. The tumor-bearing mice were arbitrarily allocated to three groups. The volume of the tumor was estimated at regular intervals. Drug administration was started when the tumor volume was about 100 mm3. Full ultrasonic dissolution of the jaceosidin was dissolved into a uniform suspension (1% DMSO + 5% CMC-Na + normal saline) and intraperitoneal injected into mice (200 μL/mouse). The body weight and the tumor volumes were recorded every day. After 7 days of continuous administration, the mice were dissected and the tumors were excised. The tumor was fixed in 4% paraformaldehyde (Biosharp, China) for routine sections preparation. Routine sections were utilized for hematoxylin/eosin (HE) staining (Servicebio, China) and immunohistochemical (IHC) analysis according to the protocol [39 (link)]. The antibodies used in the IHC assay including E-cadherin (#3195, CST), ITGA2 (ab181548, Abcam), ITGB1 (ab179471, Abcam) and Vimentin (#5741, CST).
10
Immunohistochemical Analysis of EMT Markers
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Tumor tissues were fixed in formalin and embedded in paraffin. Sections (5-µm thick) were cut from the embedded tissues and mounted on polylysinecoated slides. Tumor sections were subjected to IHC staining for detection of E-cadherin, VIM and ITGB1. Briefly, sections were deparaffinized in xylene, rehydrated in gradient alcohol and treated with 0.3% H 2 O 2 for 15 min to quench endogenous peroxidase activity. Following antigen retrieval, sections were blocked in 10% normal serum with 1% BSA in TBS for 2 h at room temperature, and then incubated at 4˚C overnight with the corresponding primary antibodies (E-cadherin, vIM and ITGB1; Abcam). Negative controls were incubated with the negative control antibody under the same condition. Next, the sections were incubated with biotinylated secondary antibody for 1 h, followed by incubation with conjugated horseradish peroxidase streptavidin for 1 h. Finally, the sections were incubated with diaminobenzidine and counterstained with hematoxylin.
Statistical analysis. Experiments were performed at least three times. Data were analyzed by Student's t-test when comparing two groups and by one-way ANOvA followed Bonferroni's post-test, when comparing more than two groups. A P-value <0.05 was considered to indicate a statistically significant result.
Statistical analysis. Experiments were performed at least three times. Data were analyzed by Student's t-test when comparing two groups and by one-way ANOvA followed Bonferroni's post-test, when comparing more than two groups. A P-value <0.05 was considered to indicate a statistically significant result.
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