Total proteins were extracted from knee cartilage tissues using the RIPA lysate (Meilunbio, Dalian, China). Protein samples were loaded onto SDS-PAGE gel (Beyotime, Shanghai, China) for electrophoresis and transferred onto a PVDF membrane. After immersing in 5% skim milk for 2 h, membranes in interested sizes were incubated with the primary antibodies of anti-β-actin (1:1000, Sc-47,778, Santa Cruz Biotechnology, TX, U.S.A.), anti-MMP-13 (1:1000, 18165-1-AP, Proteintech, CA, U.S.A.), anti-Col-II (1:1000, 28459-1-AP, Proteintech), anti-AKT1/2/3 (1:1000, bs- 6951R, Bioss, MA, U.S.A.), anti-phospho-AKT (1:1000, bs0876R, Bioss), anti-PI3KCA (1:1000, 2067R, Bioss) and anti-PI3K p110 beta (1:1000, bs-10657R, Bioss) overnight at 4 °C. On the other day, they were incubated with HRP-linked anti-rabbit IgG (1:5000, #7074, Cell Signaling Technology, MA, U.S.A.) and HRP-linked anti-mouse IgG (1:5000, #7076, Cell Signaling Technology) at room temperature for 1 h. Images were visualized using an enhanced chemiluminescence kit (Meilunbio) and grey values were analyzed using ImageJ software.
Bs 0876r
Manufactured by Bioss Antibodies
Sourced in United States, China
The Bs-0876R is a piece of lab equipment designed for use in biological research. It is a versatile instrument that can perform a variety of tasks. The core function of the Bs-0876R is to facilitate the analysis and manipulation of biological samples. Further details about its intended use or specific capabilities are not available.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Sds page gel, by Beyotime (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions)
Hrp linked anti mouse igg, by Cell Signaling Technology (1 mentions)
Ripa lysate, by Meilun (1 mentions)
Bs 6951r, by Bioss Antibodies (1 mentions)
Enhanced chemiluminescence kit, by Meilun (1 mentions)
P akt, by Bioss Antibodies (1 mentions)
P pi3k, by Cell Signaling Technology (1 mentions)
Wbkls0100, by Merck Group (1 mentions)
Fd006, by Fdbio (1 mentions)
Af6265, by Affinity Biosciences (1 mentions)
Ac033, by ABclonal (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Af5228, by Affinity Biosciences (1 mentions)
Mouse il 6 elisa kit, by Boster Bio (1 mentions)
Lipofectaminetm 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Primescripttm rt reagent kit with gdna eraser perfect real time, by Takara Bio (1 mentions)
4 protocols using bs 0876r
1
Cartilage Protein Profiling by Western Blot
2
Western Blot Analysis of AKT/PI3K Pathway
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Wound tissues were lysed with RIPA buffer and total protein was extracted and quantified using the BCA kit (Thermo, USA). After protein denaturation at 100 °C for 10 min, an equal volume of loading buffer was added to the sample before SDS-PAGE electrophoresis. Proteins were then transferred to a PVDF membrane and blocked with 5% skim milk at room temperature for 1 h with gently shaking. Membranes were incubated with the following primary antibodies; AKT (1:1000, 4691S, Cell Signaling Technology), p-AKT (1:300, bs-0876R, Bioss), PI3K (1:500, 4292S, Cell Signaling Technology), P-PI3K (1:300,17366S, Cell Signaling Technology), or β-actin (1:5000, G1001, GXY bioSci & Tech Co., Ltd.) overnight at 4 °C. Afterward, Anti-Rabbit IgG (H + L) Antibody, DyLight™ 680-Labeled (1:5000, 5230-0402, SeraCare) was incubated at room temperature for 1 h. Protein bands were visualized using the far-infrared laser imaging system (LI-COR, USA). Gray value intensities of the bands were quantified using the Image J software.
3
Protein Extraction and Western Blot Analysis
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The proteins were extracted from the samples using the radio immunoprecipitation assay lysis buffer (P0013B, Beyotime, China), containing a protease-inhibitor cocktail (HY-K0010, MCE, USA). The proteins were lysed in SDS-loading buffer (FD006, Fdbio, China), then the lysates were resolved on sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane (IPVH00010, Millipore, USA). The membrane was incubated with polyclonal antibodies against EGFR (AF6043, Affinity, China), phospho-EGFR (AF3047, Affinity, China), MMP2 (AF0577, Affinity, China), MMP9 (AF5228-MMP-9+Antibody.html">AF5228 , Affinity, China), VE-Cadherin (AF6265, Affinity, China), AKT (bsm-33282M, Bioss, China), phospho-AKT (bs-0876R, Bioss, China), Foxq1 (PA1-31951, Invitrogen, USA) or GAPDH (AC033, Abclonal, China) at a dilution of 1:1000, then incubated with species-specific HRP-conjugated secondary antibodies at a dilution of 1:5000. The immunoreactive bands were visualized by enhanced chemiluminescence (WBKLS0100, Millipore, USA). phosphatase inhibitor cocktail (HY-K0021, MCE, USA) was used for the phosphoprotein blots, e.g., p-EGFR and p-AKT.
4
Immune Response Modulation Assay Protocol
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PrimeScriptTM RT reagent kit with gDNA Eraser (Perfect Real Time) (Code No.: RR047A)and SYBR® Premix Ex TaqTM II (Tli RNaseH Plus) (Code No.: RR820A) were purchased from TaKaRa. BCA protein assay kit (KGP902) and ECL detection kit were purchased from KeyGENE (Nanjing, China). Cell culture dishes were purchased from Guangzhou Jet Bio-Filtration Co., Ltd. (Guangzhou, China). Antibodies against β-actin (60008-1-lg, mouse), interleukin (IL)-6 (21865-1-AP, rabbit), tumor necrosis factor alpha (TNF-α; 60291-1-lg, mouse), protein kinase B (AKT; 10176-1-AP, rabbit), phosphoinositide 3-kinase (PI3K; 60225-1-lg, mouse), inducible nitric oxide synthase (iNOS; 18985-1-AP, rabbit), arginase-1 (ARG1; 16001-1-AP, rabbit), SRC (11097-1-AP, rabbit), and IL-10 (20850-1-AP, rabbit) were obtained from Proteintech (Wuhan, China). Antibodies against IL-1β (bs-20448R, rabbit), IL-12 (bs-1789R, rabbit), and P-AKT (bs-0876R, rabbit) were purchased from Bioss (Beijing, China). Antibody against P-PI3K (4228, rabbit) was purchased from CST. Dasatinib inhibitor was obtained from MCE (New Jersey, United States). lipofectamineTM 3000 Reagent was purchased from Thermo ScientificTM (United States). Mouse IL-6 ELISA kit was purchased from Boster (Wuhan, China).
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