Western blots were performed with first antibodies listed below; p-Akt (#4060, Cell Signaling), Akt (#9272, Cell Signaling), p-Erk1/2 (#4370, Cell Signaling), Erk1/2 (#4695, Cell Signaling), p-IκBα (#9246, Cell Signaling), IκBα (sc-371, Santa Cruz), SYK (sc-1240, Santa Cruz), PLCγ1 (#5690, Cell Signaling), PIK3CD (sc-7176, Santa Cruz), RasGRP3 (#3334, Cell Signaling), PKCβI (sc-209, Santa Cruz), PKCβII (sc-210, Santa Cruz), IKKβ (sc-8014, Santa Cruz), MYD88 (#4283, Cell Signaling), NIK (#4994, Cell Signaling, protein was visualized by MG132 pretreatment.), FLAG (F3165, SIGMA), β-actin (sc-69879, Santa Cruz), p-RPS6 (#4858, Cell Signaling), RPS6 (#2317, Cell Signaling), RPL19 (sc-1000830, Santa Cruz), Ago2 (RN005M, MBL), GW182 (RN033P, MBL). Ras activation was assayed by comparing the amount of Ras-GTP and total Ras in identical cell lysates. The Ras-GTP was collected by pull-down with GST-Raf (Ras binding domain, Jena Bioscience). Alkaline phosphatase-conjugated anti-mouse and anti-rabbit secondary antibodies were from Promega. The blots were detected by BCIP/NBT substrate (Promega).
Pkcβi
Manufactured by Santa Cruz Biotechnology
Sourced in United States
PKCβI is a recombinant protein that represents the beta I isoform of protein kinase C. Protein kinase C is a family of serine/threonine-specific protein kinases involved in a variety of cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Pkcβii, by Santa Cruz Biotechnology (3 mentions)
Myd88, by Cell Signaling Technology (1 mentions)
Plcγ1, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Pik3cd, by Santa Cruz Biotechnology (1 mentions)
Rpl19, by Santa Cruz Biotechnology (1 mentions)
Nbt bcip substrate, by Promega (1 mentions)
P rps6, by Cell Signaling Technology (1 mentions)
Rasgrp3, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Phosphorylated erk1 2 p erk1 2, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti rabbit and anti mouse secondary antibodies, by Promega (1 mentions)
X omat 2000, by Kodak (1 mentions)
P stat3 y705, by Cell Signaling Technology (1 mentions)
Actin, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
7 protocols using pkcβi
1
Immunoblotting Analysis of Key Signaling Proteins
2
Western Blot Analysis of Protein Signaling
3
Mangiferin Characterization and Antibody Sourcing
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Mangiferin was purchased from the Innochem (Beijing, China). The purity was measured by HPLC-MS: 97.5%. The structure of mangiferin was determined by 1H NMR and MS spectrum. 1H NMR (400 MHz, DMSO-d6) δ 13.76 (s, 1H), 10.68 (s, 1H), 10.57 (s, 1H), 9.79 (s, 1H), 7.38 (s, 1H), 6.87 (s, 1H), 6.38 (s, 1H), 5.06–4.23 (m, 1H), 4.05 (t, J = 9.1 Hz, 1H), 3.75–3.65 (m, 1H), 3.41 (dd, J = 11.8, 5.8 Hz, 1H), 3.26–3.09 (m, 3H), ESI MS [M+Na]+ = 445.3.
AQP2, α-SMA, NLRP3, p-JNK, JNK, and OAT1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), URAT1, AQP1, p-PKCβI (Thr 641), and PKCβI antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). GLUT9 antibody was purchased from Abcam (Cambridge, MA, USA). Commercial kits for detecting uric acid, creatinine, BUN, SOD, and XO were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). RIPA buffer, ECL reactions were purchased from Applying Technologies Inc. (Beijing, China).
AQP2, α-SMA, NLRP3, p-JNK, JNK, and OAT1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), URAT1, AQP1, p-PKCβI (Thr 641), and PKCβI antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). GLUT9 antibody was purchased from Abcam (Cambridge, MA, USA). Commercial kits for detecting uric acid, creatinine, BUN, SOD, and XO were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). RIPA buffer, ECL reactions were purchased from Applying Technologies Inc. (Beijing, China).
4
Western Blot Analysis of TGF-β1 and PKCβI
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Cells were resuspended in lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 30 min and sonicated for 2 min at 20 W, followed by centrifugation at 12,000 × g for 10 min at 4°C. The supernatant was collected and 50 µg/lane protein (concentration determined using the bicinchoninic assay kit (Thermo Fisher Scientific, Inc.) was separated using SDS-PAGE on a 10% gel (Bio-Rad Laboratories, Inc.) and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc.), which was blocked in Tris-buffered saline/Tween-20 (TBST) with 5% non-fat milk for 1 h at 37°C. The membrane was subsequently incubated with primary antibodies against TGF-β1 (cat no. sc-146; 1:2,000), PKCβI (cat no. sc- 209; 1:1,000) and GAPDH (cat no. sc-25778; 1:500) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Following washing with TBST, the membranes were incubated with a horseradish peroxidase-conjugated labeled goat anti-rabbit secondary antibody (cat no. sc-2004; 1:500; Santa Cruz Biotechnology, Inc.) for 1 h at 4°C, followed by additional three washes with TBST. Protein bands were visualized by enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA). The Scion Image system version 4.03 (National Institutes of Health, Bethesda, MD, USA) was used to quantify band intensity and data are expressed as the mean of three independent experiments.
5
Elucidating IGF-II Signaling Pathways in Senescence
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IGF-II signal transduction may occur through different pathways (Figure 5a ). We determined which of these could be associated with senescence. In particular, the MSC cultures were incubated at 37°C for 30 min with each of the following drugs, separately: 100 nM Pertussis toxin (PTX, BMLG101-0050 Enzo Biochem, NY, USA) for the Gαi kinase, 1 μM YM-254890 (YM, 10–1590 Focus Biomolecules, PA, USA) to block Gαq/11, 50 μM D609 (sc201403, Santa Cruz Biotechology, CA, USA) to inhibit PLCβ, 10 μM BAPTA-AM (BPT) (15551 Cayman, MI, USA) and 10 mM 1,2,3,4-tetrahydrostaurosporine (STP, ab143861, Abcam, UK) for PKCα kinase inhibition, 5 nM PKCβi (Santa Cruz Biotech, TX, USA) for PKCβ inhibition, and 100 mM chlorpromazine (CPZ, sc-357313, Santa Cruz) to block endocytosis. Subsequently, we added 25 ng/ml IGF-II, and the samples were further incubated for 24 hr, and the senescence assays were performed. For each drug, we evaluated the inhibitory effect at the concentration we used (Figure 7—figure supplement 1 ).
6
Immunohistochemical Protein Localization
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Detection of protein localization was performed as previously described [11 (link)–13 (link)]. Paraffin-embedded kidney sections were cut to 4 μm in thickness. The slides were deparaffinized and endogenous peroxidase was blocked by treatment with 3% H2O2. The sections were incubated with the primary antibody to PKCβI (1 : 400; Santa Cruz Biotechnology), PKCβII (1 : 400; Abcam), NHE1 (1 : 100; Santa Cruz Biotechnology), or NHE3 (1 : 200; Santa Cruz Biotechnology) at 4°C overnight, followed by the respective horseradish peroxidase-linked secondary antibody (Bio-Rad), and then reacted with 3,3′-diaminobenzidine (DAB) solution (Sigma). As a negative control, the primary antibody was omitted, resulting in negative staining. In a blinded manner, three pathologists independently scored the staining intensity on a semiquantitative five-tiered grading scale from 0 to 4 (0 = negative; 1 = trace; 2 = weak; 3 = moderate; and 4 = strong) as previously described [11 (link)–13 (link), 19 (link)].
7
Exploring Platelet Redox Signaling
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RA was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), and Trolox was purchased from Tocris Bioscience (Ellisville, MO, USA). The following commercial antibodies were used: PKC-α, PKC-βI, PKC-βII, PKC-γ, PKC-δ (Santa Cruz Biotechnology, CA, USA), and β-actin (Cell Signaling Technology, Inc., Danvers, MA, USA). VAS2870 was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Gö6976, rottlerin, and lucigenin were purchased from Sigma-Aldrich. Eptifibatide was purchased from Millipore (Burlington, MA, USA). CellTrackerTM Green 5-chloromethylfluorescein diacetate (CMFDA), 2,7-dichlorodihydro fluorescent diacetate (H2DCFDA), and dihydroethidium (DHE) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fluorescein isothiocyanate (FITC) anti-human CD41a (integrin αIIb) antibody and APC anti-human CD61 (integrin β3) antibodies were purchased from BD Pharmingen (San Diego, CA, USA). All other chemical reagents were purchased from Sigma-Aldrich and were of analytical or high-performance liquid chromatography (HPLC) grade.
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