Luteolin (purity ≥ 98%) was purchased from Sichuan Weikeqi Biological Technology CO., Ltd (CAS#: 491‐70‐3). BMS‐5 (purity ≥ 98%) was purchased from Enzo Life Sciences (CAS#:1338247‐35‐0). CNBr‐activated SepharoseTM 4B (Lot# 10265330) was purchased from GE Healthcare. Thiazolyl blue tetrazolium bromide (MTT) powder was purchased from Solarbio Technology Co., Ltd. Dimethylsulfoxide (DMSO) was purchased from Sigma. Cyclin D1 (catalog# 2922), cyclin D3 (catalog# 2936), Bax (catalog# 5023), cleaved‐PARP (catalog# 5625), caspase‐3 (catalog# 9662), cleaved caspase‐3 (catalog# 9664), caspase‐7 (catalog# 9492), cleaved caspase‐7 (catalog# 8438), ROCK1 (catalog# 4035) and ROCK2 (catalog# 9029) were purchased from Cell Signaling Technology. Phospho‐LIMK‐1/2 (Thr 508/505)‐R (catalog# sc‐28409‐R), LIMK‐1(catalog# sc‐28370), cofilin (catalog# sc‐33779) and phospho‐cofilin (mSer3)‐R (catalog# sc‐21867‐R) were purchased from Santa Cruz Technology.
Rock2
Manufactured by Cell Signaling Technology
Sourced in United States
ROCK2 is a laboratory product offered by Cell Signaling Technology. It is a protein that plays a key role in cellular signaling pathways. ROCK2 is a member of the Rho-associated protein kinase family and is involved in the regulation of various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Rock1, by Cell Signaling Technology (5 mentions)
P stat3, by Cell Signaling Technology (3 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
Py397 fak, by Thermo Fisher Scientific (2 mentions)
Collagen 3, by Abcam (2 mentions)
Total stat3, by Cell Signaling Technology (2 mentions)
Fibronectin, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Integrin β1, by Merck Group (2 mentions)
P mlc2, by Cell Signaling Technology (2 mentions)
Tenascin c, by Abcam (2 mentions)
Gli 1, by Thermo Fisher Scientific (2 mentions)
Collagen 12a1, by Santa Cruz Biotechnology (2 mentions)
Mabt409, by Thermo Fisher Scientific (2 mentions)
Total fak, by BD (2 mentions)
P mypt1, by Merck Group (2 mentions)
P smad2 3, by Cell Signaling Technology (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
Nlrp3, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
12 protocols using rock2
1
Luteolin and BMS-5 Cellular Assays
2
Immunohistochemistry Antibody Panel
3
Osteoclast Differentiation Assay
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The fetal bovine serum (FBS) and α-MEM were provided by Gibco (California, USA). M-CSF and RANKLwere purchased from R&D (MN, USA). The tartrate-resistant acid phosphatase (TRAP) staining kit was purchased from Sigma-Aldrich (St Louis, MO, USA). HiPerFect Transfection Reagent was purchased from Qiagen (GmbH, Hilden, Germany). SC-79 was obtained from MedChemExpress (New Jersey, USA). Trap5b and CTX-I Elisa kit were purchased from Elabscience (Wuhan, China). Phalloidin-iFluor 488 (ab176753) was purchased from Abcam (Cambridge, UK). Antibodies for NFATc1 (#64,602) was from Biolegend (San Diego, CA). S6 (#2217), phospho-S6 (#4858), p70S6K (#9202), phospho-p70S6K (#9204), Akt (#4685), phospho-Akt (#31957), mTOR (#2983) and Rock2 (#47012) antibodies were from Cell Signaling Technology (Danvers, MA, USA). RhoA (ab187027) and GAPDH (ab8245) antibodies were from Abcam (Cambridge, UK). Flag (AF0036) antibody was from Beyotime Biotechnology (Shanghai, China).
4
Western Blot Analysis of EMT Markers
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Cells were lysed with RIPA buffers (Thermo Fisher Scientific), and equal amounts of protein for each sample were separated by 10% SDS/PAGE and transferred to the polyvinylidene fluoride membrane (Sangon Biotech, Shanghai, China). The membranes were incubated with primary antibodies, including E‐cadherin (1 : 1000, Cell Signaling Technology, Boston, MA, USA), Vimentin (1 : 1000; Cell Signaling Technology), ROCK2 (1 : 1000; Cell Signaling Technology), and GAPDH (1 : 1000; Absin Bioscience Inc., Shanghai, China), respectively, at 4 °C overnight. Membranes were washed and incubated with the HRP‐conjugated goat anti‐mouse IgG (1 : 1000; Beyotime, Shanghai, China) or goat anti‐rabbit IgG (1 : 5000; Abcam) for 2 h. The protein bands were detected by ECL (Thermo Fisher Scientific).
5
Immunohistochemistry Antibody Panel
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Antibodies were as follows: a-SMA (Sigma-Aldrich C6198, 1:1000), GLi-1 (Thermo Scientific PA5-32206, 1:500), Tenascin C (Abcam AB108930, 1:500), Fibronectin (Abcam AB2413, 1:500), Collagen 12A1 (Santa Cruz Biotechnology E-15 sc-68449, 1:200), Sox2 (Abcam ab97959, 1:500), Vimentin (Cell Signaling 5741S, 1:200), FAP (Abcam AB53066, 1:500), Collagen III (Abcam AB7778, 1:500), YAP (Cell signaling 4912, 1:200), β1integrin (EMD Millipore MABT409, 1:500), pY397-FAK (Invitrogen 44625, 1:200), total FAK (BD Biosciences 610088, 1:1,000), ROCK1 (Cell Signaling C8F7, 1:1,000), ROCK2 (Cell Signaling D1B1, 1:1,000), p-MLC2 (Cell Signaling 3671, 1:200), p-MyPT1 (Millipore ABS45, 1:200), p-Stat3 (Cell Signaling 9145, 1:200), p-SMAD2/3 (Cell Signaling 8828, 1:200), total Stat3 (Cell Signaling 9132, 1:1,000), GAPDH (Cell Signaling 2118, 1:5,000), CD45 (BD Biosciences 550539, 1:200), CD68 (Thermo Scientific Ab-3, 1:200), Alexa Fluor-conjugated goat secondary anti–mouse IgG and anti–rabbit IgG antibodies (Invitrogen A11012, 1:1,000 and A11005, 1:1,000) and HRP-conjugated rabbit secondary antibody (GE health care life sciences NA934VS, 1:5,000).
6
Western Blot Analysis of Neuroinflammatory Signaling
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Western blot analyses were performed according to standard protocol [49 (link)] using the following antibodies against: α7-nAChR (ab216485; 1 : 1000), p38 (ab31828; 1 : 1000), p-p38 (phospho T180+Y182) (ab4822; 1 : 1000), IL-6 (ab9324; 1 : 1000), inducible NO synthase (ab3523; 1 : 1000), and GAPDH (ab9484; 1 : 10,000), purchased from Abcam (Abcam plc., Cambridge, UK), and RhoA (#2117; 1 : 1000), RhoA-GTP (#8820; 1 : 1000), ROCK1 (#4035; 1 : 1000), ROCK2 (#9029; 1 : 1000), t-MBS (#2634; 1 : 1000), and p-MBS (#3040; 1 : 1000) from Cell Signaling Technology (Cell Signaling, Danvers, MA, USA) in Supplementary Table S1 . The protein bands were pictured using enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, NJ, USA) and quantified using ImageJ software (https://imagej.nih.gov/ij/ ).
7
Myogenic Marker Expression Profiling
8
Protein Expression Analysis in HPMVECs
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Cultured HPMVECs were homogenized with the cell lysis buffer system (Santa Cruz) on dry ice. The protein was extracted using protein extraction reagents (Beyotime) per the manufacturer’s instructions. After centrifugation of homogenates, the concentration of protein was measured with BCA method by using a BCA assay kit (Invitrogen). Protein samples were separated by SDS-PAGE and then transferred to the PVDF membranes electronically. The membranes were then incubated with the blocking buffer (Santa Cruz) to eliminate the non-specific binding sites. Then, the membranes were incubated with primary antibodies against ROCK2 (Cell Signaling Tech, 1: 2000), eNOS, (Cell Signaling Tech, 1: 2000) p-eNOS (Cell Signaling Tech, 1: 2000), Bcl2 (Santa Cruz, 1: 2000), active caspase3 (Abcam, 1: 2000), NF-κB (Abcam, 1: 1000), IL-6 (Abcam, 1: 2000), TNF-α (Abcam, 1: 1000), GAPDH (Abcam, 1: 5000), and Histone-H3 (Abcam, 1: 2000) at 4°C for 12 h. After washing in TBST, membranes were further incubated with secondary antibodies conjugated with horseradish peroxidase (Abcam) at room temperature for 2 h. The immunoblots were visualized with an enhanced chemiluminescence Western blotting detection system (ECL, Fisher Scientific).
9
Antibody and inhibitor sources for SIK2, MYLK, and ROCK
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Primary antibodies against SIK2, F‐actin, MYL2, phospho‐MYL2, ROCK1 and ROCK2 were purchased from Cell Signaling Technology. MYLK antibody, SIK2‐pS358 antibody and GAPDH antibody were purchased from Abcam (Cambridge, MA, USA), Kinexus (Vancouver, BC, Canada) and Sigma‐Aldrich (St. Louis, MO, USA), respectively. SIK2 inhibitor, ARN‐3236, was purchased from Selleckchem (Houston, TX, USA), while MYLK inhibitor, ML‐9, was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Human gateway entry clone empty vector and MYLK (Human Gene and Protein Database clone number: MYLK) were obtained from Shanghai Integrated Biotech Solutions Company (Shanghai, China).
10
Signaling Pathway Regulation in Cell Culture
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Antibodies were purchased from the following sources: p-EGFR, EGFR, p-ERK1/2, ERK1/2, p-STAT3, STAT3, p-Src, Src, COX-2, eNOS, HIPK-2, ATF3, NLRP3, and ROCK2 were purchased from Cell Signaling Technology (Dancers, MA); The secondary antibodies for immunoblot analysis were from Jackson Immunoresearch (West Grove, PA). LPS was purchased from Sigma (St. Louis, MO). Gefitinib, dasatinib, U0126, and S3I-201 were purchased from AstraZeneca (Macclesfield, England).
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