Proteins were extracted in RIPA lysis buffer (P0013B, Beyotime) and the concentration was determined using a BCA Protein assay kit (P0010S, Beyotime). Proteins were separated on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk and then incubated overnight with primary antibodies, including anti-CAP3 (1:1000, ARG58412, and Arigo Biolaboratories Corp.), anti-S100P (1:1000, 7677S, and CST), anti-PTTG1 (1:1000, 13445, and CST), anti-LOXL2 (1:1000, ab96233, and Abcam), anti-MELTF (1:1000, 10428-1-AP, and Proteintech), anti-PKP2 (1:1000, ab189323, and Abcam), anti-TMPRSS11E (1:1000, PA5-48775, and Invitrogen Antibodies), and anti-β-actin (1:10000, A5441, and Sigma). Horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG secondary antibody was used to incubate membranes at room temperature. The blots were detected by chemiluminescence and imaged on an AlphaView analysis system (ProteinSimle, USA). The quantification of individual protein bands was assessed by densitometry using ImageJ software.
Ab96233
Manufactured by Abcam
Sourced in United Kingdom
Ab96233 is a laboratory device designed for the detection and quantification of specific molecules or proteins in a sample. It utilizes an array-based approach, but further details about its technical specifications or intended applications are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Mab374, by Merck Group (2 mentions)
Sc 5293, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ab189323, by Abcam (1 mentions)
A5441, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Clarity western substrate, by Bio-Rad (1 mentions)
Anti rabbit igg horseradish peroxidase hrp linked antibodies, by Cell Signaling Technology (1 mentions)
Ab8245, by Abcam (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Echochemiluminescence ecl detection system, by GE Healthcare (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Ab174316, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica camera (1 mentions)
Ab137867, by Abcam (1 mentions)
Ab216842, by Abcam (1 mentions)
12 protocols using ab96233
1
Protein Expression Analysis by Western Blot
2
Immunoblotting Analysis of LOXL2 and PLOD2
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Cells were harvested 72 h after transfection, and lysates were prepared. Protein lysates (20 μg) were separated on Mini-PROTEAN TGX gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to PVDF membranes. Immunoblotting was performed with rabbit anti-LOXL2 antibodies (1:1000; ab96233; Abcam, Cambridge, UK) and rabbit anti-PLOD2 antibodies (1:300; 21214-1-AP; Proteintech Group, Inc., Chicago, IL, USA). Anti-GAPDH antibodies (1:1,000; ab8245; Abcam) were used as an internal loading control. The membranes were washed and incubated with anti-rabbit IgG horseradish peroxidase (HRP)-linked antibodies (#7074; Cell Signaling Technology). Complexes were visualized with Clarity Western Substrate (Bio-Rad Laboratories).
3
Western Blot Analysis of LOXL2 Protein
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After 96 h of transfection, protein lysates (50 μg) were separated on NuPAGE on 4–12% Bis-Tris gels (Invitrogen) and transferred to polyvinylidene fluoride membranes. Immunoblotting was performed with diluted anti-LOXL2 antibodies (1:1,000; ab96233; Abcam, Cambridge, UK) and anti-GAPDH antibodies (1:5,000; MAB374; Chemicon, Temecula, CA, USA). The membrane was washed and then incubated with an anti-rabbit-IgG, HRP-linked antibody (#7074; Cell Signaling Technology, Danvers, MA, USA). Specific complexes were visualized with an echochemiluminescence (ECL) detection system (GE Healthcare, Little Chalfont, UK) as described previously (9 (link)–11 (link)).
4
Immunocytochemistry of LOX and LOXL2
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HTR-8/SVneo cells were seeded in glass-bottom cell culture dishes and grown to 70% confluence. Then, the cells were fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Following washing in PBS and blocking by incubation in 5% (w/v) bovine serum albumin (BSA) in PBS (buffer A), cells were incubated overnight with anti-LOX (ab174316, 1:100, Abcam, Cambridge, MA, USA) and anti-LOXL2 (ab96233, 1:100, Abcam) antibodies diluted in buffer A. Following washing, the cells were incubated for 1 h in Alexa Fluor 488 goat anti-rabbit secondary antibodies (A11070, 1:200 for LOX and 1:1000 for LOXL2, Invitrogen, Carlsbad, CA, USA) diluted in buffer A. The nuclei were stained with 4′,6-diamidino-2-phenylindole. Fluorescence images were obtained using confocal microscopy (TCS SP8; Leica,Wetzlar, Germany).
5
Western Blot Analysis of Protein Markers
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Protein level in lysis supernatant of BxPc‐3 cells was determined by bicinchoninic acid (BCA) protein assay kit (Thermo), and 25 μg of which was separated on 10% to 15% of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Electrophoretic pure of HIF‐1α, LOXL2, E‐cadherin, MMP2, and MMP9 were transferred onto nitrocellulose (NC) membranes (Millipore), and incubated with antibody against HIF‐1α (abcam, Ab216842), anti‐LOXL2 antibody (abcam, Ab96233), antibody against E‐cadherin (Cell Signaling Technology [CST], #14472), antibody against MMP2 (Abcam, Ab14311), antibody against MMP9 (Abcam, Ab137867), anti‐β‐actin antibody (Abcam, ab8226), and anti‐GAPDH antibody (CST, #5174) at 4°C overnight followed by horseradish peroxidase‐conjugated antibodies (Beyotime, A0208, Shanghai, China) for another 1 hour at 25°C. Immunoreactive bands were qualified by enhanced chemiluminescence (ECL) system (GE Healthcare/Amersham Biosciences). We chose β‐actin as a “loading” control (instead of GAPDH) in hypoxic studies, accordingly to a reported study.19
6
Immunoblotting Analysis of Fibrosis Markers
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The blots were reacted with antibodies of anti-LOXL2 (1:500, ab96233, Abcam), anti-TGF-β2 (1:1,000, MAB73461, R&D Systems), anti-GAPDH (1:20,000, G9545, Sigma-Aldrich), anti-TFIIb (1:1,000, ab109106, Abcam), anti- phosphor-SMAD2 (1:1,000, 138D4, Cell Signaling), anti-SMAD2 (1:1,000, D43B4, Cell Signaling), anti-GAPDH (1:1,000, 2118, Cell Signaling), α–SMA (1:1,000, A2547, Sigma-Aldrich), total AKT (1:1,000, 4685, Cell Signaling), phospho-AKT (Ser473) (1:1,000, 4060, Cell Signaling), phospho-AKT (Thr308; 1:1,000, 4056, Cell Signaling), phospho-S6K1 (1:1,000, 9234, Cell Signaling), total S6K1(1:1,000, 9202, Cell Signaling), phospho-4EBP1 (1:1,000, 2855, Cell Signaling), PI3Kα (1:1,000, C73FB, Cell Signaling), PI3Kβ (1:1,000, C33D4, Cell Signaling) and PI3Kδ (1:1,000, D55D5, Cell Signaling). Then followed by HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Chemiluminescence was detected with ECL Western blot detection kits (GE) with LI-COR odyssey image system. Uncropped blot images are shown in Supplementary Fig. 7 .
7
LOXL2 Protein Expression and Localization in Tumors
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To determine the protein expression and cellular localization of LOXL2, a 4 μm section of each tumor specimen was subjected to IHC analysis. The slides were deparaffinized, rehydrated, and treated with 3% H2O2 in methanol for 15 min to inhibit endogenous peroxidase. After blocking with 10% nonimmunogenic goat serum at room temperature for 30 min, slides were incubated with rabbit anti-LOXL2 polyclonal antibody (ab96233, Abcam, Cambridge, UK) overnight at 4°C. PBS incubation was parallelly conducted as the negative control. Immunoreactivity was assessed with a 2-step assay kit (PV-6001, ZSGB-Bio, Beijing, People’s Republic of China) and a DAB IHC detection kit (ZAI9017, ZSGB-Bio) according to the manufacturer’s instructions. After counterstaining, dehydration, and mounting, 2 pathologists blinded to the clinical data were invited to evaluate the IHC results based on both staining proportion and intensity. The positive proportion was scored as 0 (0%–10%), 1 (11%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (75%–100%); the staining intensity was scored as 0 (negative), 1 (light yellow, weak), 2 (yellow, moderate), and 3 (dark brown, strong). The final IHC score was calculated by multiplying the 2 scores above. IHC score <8 was considered as low expression, while IHC score ≥8 was grouped into high expression.
8
Protein Concentration and Analysis
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Secreted proteins were first concentrated using Amicon Ultra Cel-10 centrifugal filters (MWCO 10.000; Millipore, Bedford, MA, USA) as previously described [94 (link)]. Cellular lysates or secreted proteins were resolved by 10% SDS-PAGE, and the proteins were electrophoretically transferred onto nitrocellulose membrane (Bio-Rad, Herwles, Hercules, CA, USA) and analyzed as previously [24 (link)]. Secreted and cellular protein bands of human melanoma cells and fibroblasts were quantified using Image Studio Lite (LI-COR Biotechnology, Lincoln, NE, USA). A rabbit polyclonal antibody recognizing the C-terminal domain of LOX (L4669; Sigma-Aldrich), a rabbit polyclonal LOX propeptide antibody to LOX-PP (NB110-41568; Novus Biologicals, Littleton, CO, USA), and a rabbit polyclonal antibody to LOXL2 (ab96233; Abcam, Cambridge, UK) were used to detect the respective proteins. Secreted protein samples were verified to contain comparable amounts of total proteins by silver staining. Mouse monoclonal antibodies to alpha-tubulin (DM1A; Abcam) and actin (JLA20; Merck, Millipore, Billerica, MA, USA) were used as loading controls for cellular proteins.
9
Western Blot Analysis of LOX and LOXL2
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Proteins were extracted with RIPA buffer containing protease inhibitors, and concentration was determined using the BCA assay (Pierce). Whole-cell extracts were resolved by SDS-PAGE, probed with a specific primary antibody, and revealed with the enhanced chemiluminescence system (Pierce). Quantifications were made using the Quantity One software (Bio-Rad). Antibodies used were as follow: LOX (ab3128, 1/1,000 dilution, Abcam); LOXL2 (ab96233, 1:1,000 dilution, Abcam); tubulin (T5168, 1:2,000 dilution, Sigma); HRP-conjugated secondary goat anti-mouse (1:2,000 dilution, BioRad), and HRP-conjugated secondary goat anti-rabbit (1:2,500 dilution, BioRad).
10
Protein Isolation and Quantification for Western Blot
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The total protein was isolated from the cells using RIPA lysis buffer (Beyotime, Shanghai, China) and protein concentration was quantified using a BCA kit (Beyotime, Shanghai, China) [17 (link)]. SDS-PAGE was used to dissolve the protein samples (30 μg/lane), and then the protein was transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Then, the PVDF membranes were blocked with 5% skim milk at room temperature for 1 h, and incubated with primary antibodies including anti-LOXL2 (1:1000, ab96233, Abcam Inc., Cambridge, UK), anti-GAPDH (1:1000, ab9485, Abcam Inc., Cambridge, UK) at 4°C overnight. Next, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:2000, ab150077, Abcam Inc., Cambridge, UK) for 1 h at room temperature, and finally, the protein bands were detected using an ECL kit (ThermoFisher Scientific, Waltham, MA, USA).
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