NSCs were incubated with 0.5 nM fNCs at predetermined time points. Then, cells were washed with ice cold PBS (calcium and magnesium free) three times before being treated with RIPA buffer (Pirece) with protease inhibitor cocktail (Roche) for 10 min on ice. Cell lysates were harvested and subjected to centrifugation at 13 000 rpm at 4 °C for 10 min. The resultant supernatants containing protein were harvested, followed by 4–12% gradient SDS-PAGE gel separation. Nitrocellulose membranes were used for transferring separated protein bands. The membranes were blocked by 5% skim milk for 1 h at room temperature and then immunoblotted with anti βIII tubulin (1:1000) and anti NeuN (1:1000) primary monoclonal antibodies (Cell Signaling Technology, Inc.). HRP conjugated goat anti-rabbit secondary antibody (1:3000) was used to visualize immunoreactive bands. Anti β-actin rabbit mAb (Cell Signaling Technology, Inc.) (1:2000) was used as the housekeeping control.
Anti β actin rabbit mab
Manufactured by Cell Signaling Technology
Sourced in Japan
Anti-β-actin rabbit MAb is a monoclonal antibody that recognizes the β-actin protein. It is a useful tool for the detection and quantification of β-actin in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Blocking one, by Nacalai Tesque (2 mentions)
Primary monoclonal antibodies, by Cell Signaling Technology (1 mentions)
Anti β 3 tubulin, by Cell Signaling Technology (1 mentions)
Anti neun, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Chemi lumi one l, by Nacalai Tesque (1 mentions)
Bcip nbt solution kit for alkaline phosphatase stain, by Nacalai Tesque (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Total protein extraction buffer, by Beyotime (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Anti vimentin rabbit mab, by Cell Signaling Technology (1 mentions)
Anti e cadherin rabbit mab, by Cell Signaling Technology (1 mentions)
Dab substrate, by Merck Group (1 mentions)
Anti fak rabbit mab, by Cell Signaling Technology (1 mentions)
Nitrocellulose blotting membrane, by GE Healthcare (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Fusion chemiluminescence imaging system, by Vilber (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
6 protocols using anti β actin rabbit mab
1
Quantification of Neural Protein Markers
2
Immunoblot Analysis of Transfected Cells
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The gene-transfected DO-11.10 cells and silk solutions from W1, S01, S13, and S15 strains were processed for immunoblot analyses according as described previously14 (link). After blocking with Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature and incubated with anti-Myc polyclonal antibody (MBL, code no. 562, Nagoya, Japan), anti-β-actin rabbit MAb (Cell Signaling Technology, code no. #4970, Danvers, MA, USA), and anti-FibL polyclonal antibody14 (link), followed by horseradish peroxidase conjugated anti-rabbit immunoglobulins (Igs) (Dako, code no. P0399, Glostrup, Denmark), and imaged with Chemi-Lumi One L (Nacalai Tesque).
3
Western Blot Analysis of Transfected Cells
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The gene-transfected DO-11.10 cells and silk solutions from W/cs1, S01, and WS19 strains were treated with 2 × SDS sample buffer, separated using 12% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Bio-Rad). The blots were blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) for one hour at room temperature and incubated with anti-Myc polyclonal antibody (MBL, code no. 562, Nagoya, Japan), anti-β-actin rabbit MAb (Cell Signaling Technology, code no. #4970, Danvers, MA, USA), and anti-FibL polyclonal antibody (raised against a synthetic peptide representing FibL residues 67–80), followed by alkaline phosphatase-conjugated anti-rabbit immunoglobulins (Igs) (Dako, code no. D0306, Glostrup, Denmark). Immunoreactive proteins were detected using BCIP-NBT Solution Kit for Alkaline Phosphatase Stain (Nacalai Tesque).
4
Western Blot Analysis of Cell Signaling
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Cells and tissues were lysed by a total protein extraction buffer (Beyotime Biotechnology, Shanghai, People's
Republic of China). Equal amounts of lysate samples were then separated by SDS-PAGE and immunoblotted with primary antibodies and corresponding HRP-labeled secondary antibodies. Immuno-reactive protein bands were visualized in dark room after incubated with a DAB substrate (Millipore, Darmstadt, Germany). The primary and secondary antibodies employed in this work including anti-FAK Rabbit mAb (1:1,000, #71433), anti-E-Cadherin Rabbit mAb (1:1000, #3195), anti-N-Cadherin Rabbit mAb (1:1,000, #13116), anti-Vimentin Rabbit mAb (1:1000, #5741), anti-β-Actin Rabbit mAb (1:1,000, #4970) and (HRP)-linked anti-rabbit IgG antibody (1:2000, #7074), which were all obtained from Cell Signaling Technology (Massachusetts, USA).
Republic of China). Equal amounts of lysate samples were then separated by SDS-PAGE and immunoblotted with primary antibodies and corresponding HRP-labeled secondary antibodies. Immuno-reactive protein bands were visualized in dark room after incubated with a DAB substrate (Millipore, Darmstadt, Germany). The primary and secondary antibodies employed in this work including anti-FAK Rabbit mAb (1:1,000, #71433), anti-E-Cadherin Rabbit mAb (1:1000, #3195), anti-N-Cadherin Rabbit mAb (1:1,000, #13116), anti-Vimentin Rabbit mAb (1:1000, #5741), anti-β-Actin Rabbit mAb (1:1,000, #4970) and (HRP)-linked anti-rabbit IgG antibody (1:2000, #7074), which were all obtained from Cell Signaling Technology (Massachusetts, USA).
5
Western Blot Analysis of Gal-7 and STAT Proteins
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Cells were lysed in RIPA buffer (Cell Signaling Technology), and the protein was separated by 10% SDS‐PAGE (both from Thermo Fisher Scientific) and transferred onto nitrocellulose blotting membranes (GE Healthcare Life Science). After blocking, staining with primary antibody and washing, the membranes were incubated with horseradish peroxidase–conjugated goat anti‐rabbit IgG antibody. The membranes were incubated with ClarityTM Western ECL Substrate (Bio‐Rad), and the images were revealed with FUSION Chemiluminescence Imaging System (Vilber Lourmat). Intensities of the bands were quantified by ImageJ 1.46r. The following antibodies or kit were used: rabbit anti‐Gal‐7 polyclonal antibody (ab10482; Abcam), rabbit anti‐β‐actin mAb (#4970; Cell Signaling Technology), Stat Ab Sampler Kit and Phospho‐Stat Ab Sampler Kit (both from Cell Signaling Technology).
6
Quantifying Protein Expression Dynamics
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To extract total protein, the NP cells which were treated by previous steps were cleaned with cold PBS 2–3 times. Then, they lysed with RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). The concentration of the total protein was determined by the use of a BCA kit (Thermo Fisher Scientific, San Jose, CA, US). The primary antibodies were as follows: anti-p-JAK1, anti-p-JAK2, anti-p-JAK3, anti-caspase-1, anti-caspase-3, anti-p-STAT1, and anti-GAPDH. Afterwards, the transfer membrane was cleaned by TBST 3 times. Secondary antibodies combined with HRP were incubated with the transfer membrane for 1 h. A strengthened chemiluminescence assay kit (Thermo Fisher Scientific, San Jose, CA, USA) was applied to detected Protein bands. Changes in protein expression were estimated using Image J.
Rabbit anti-p-JAK1 (Tyr1034/1035) antibody (CST#3331), rabbit anti-p-JAK2 (Tyr1007/1008) antibody (CST#3771), rabbit anti-p-JAK3 (Tyr980/981) antibody (CST#5031), rabbit anti-Caspase-1 antibody (CST#2225), rabbit anti-β-Actin mAb (CST#3700), rabbit anti-p-STAT1 (Tyr701) antibody (CST # 9167), and rabbit anti-Caspase-3 antibody (CST # 9662) were purchased from Cell Signaling Technology, Inc. (Shanghai, China).
Rabbit anti-p-JAK1 (Tyr1034/1035) antibody (CST#3331), rabbit anti-p-JAK2 (Tyr1007/1008) antibody (CST#3771), rabbit anti-p-JAK3 (Tyr980/981) antibody (CST#5031), rabbit anti-Caspase-1 antibody (CST#2225), rabbit anti-β-Actin mAb (CST#3700), rabbit anti-p-STAT1 (Tyr701) antibody (CST # 9167), and rabbit anti-Caspase-3 antibody (CST # 9662) were purchased from Cell Signaling Technology, Inc. (Shanghai, China).
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