Total proteins were extracted in lysis buffer with 10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.2 mmol/L ethylene glycol-bis (β-aminoethyl ether)-N,N,N´,N´-tetraacetic acid, and 0.5% Nonidet P40 supplemented with 1 mmol/L dithiothreitol, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, and 1 mmol/L NaF. Lysates were clarified by centrifugation and separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Membranes were incubated with antibodies against anti-human FMRP, CREB, MEF2a (Cell Signaling, Danvers, MA); pRIPK3, RIPK3, pMLKL, MLKL, RIPK1 (Abcam, Cambridge, UK); pRIPK1 (SAB, Maryland, WA) (final dilution 1:1000); anti-mouse FMRP, pRIPK3, RIPK3, pMLKL, MLKL, RIPK1 (Abcam); pRIPK1 (SAB), and PARP-1 (Cell Signaling) (final dilution 1:1000), followed by a secondary antibody conjugated to HRP (Dako, Agilent Technologies). A mouse anti-β-actin antibody was used to detect β-actin and for normalization. A computer-assisted scanning densitometry was used to analyze the intensity of the immunoreactive bands.
Ripk1
Manufactured by Abcam
Sourced in United States, United Kingdom
RIPK1 is a serine/threonine protein kinase involved in cell signaling pathways. It plays a key role in the regulation of cell death and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
P ripk3, by Abcam (2 mentions)
P mlkl, by Abcam (2 mentions)
Ripk3, by Abcam (2 mentions)
Vegf c, by Santa Cruz Biotechnology (2 mentions)
Mef2a, by Cell Signaling Technology (1 mentions)
Parp1, by Cell Signaling Technology (1 mentions)
Bca kit, by Solarbio (1 mentions)
Trans blot machine, by Bio-Rad (1 mentions)
Protein extraction kit, by Solarbio (1 mentions)
Il 1β, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Annexin 5 pi kit, by Keygen Biotech (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Trypsin edta solution, by Solarbio (1 mentions)
Age bsa, by Abcam (1 mentions)
Hoechst 33258, by Beyotime (1 mentions)
D pinitol, by Merck Group (1 mentions)
6 protocols using ripk1
1
Protein Extraction and Western Blot Analysis
2
Extracting Liver Proteins and Western Blot Analysis
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Total proteins from liver tissue and primary hepatocytes were extracted using a protein extraction kit (Solarbio, Beijing, China) and measured by a BCA kit (Solarbio, Beijing, China). After heat treatment at 98 °C, the protein samples were loaded onto 12% SDS-polyacrylamide gels and electrophoresis was performed; the proteins were transferred onto nitrocellulose membranes by a Trans-Blot machine (Bio-Rad, Hercules, CA, USA), and the filters were blocked with 2.5% BSA in TBST buffer for 2 h at room temperature (RT). Then, the proteins were probed with primary antibodies for 2.5 h at RT, including anti-β-actin, -PLA2-3, -RIPK-1, -RIPK-3, -MLKL, -P-MLKL (Ser 345), -TNF-α, and -IL-1β antibodies (Abcam, USA). β-actin was used as the internal reference to ensure equal loadings. After washing with PBST buffer (3 × 8 min), the membranes were incubated with secondary antibodies for 1 h at RT and then washed with PBST buffer (4 × 10 min). Finally, the membranes were detected utilizing the ECL reagent and analyzed by Image J software (Rawak Software, Inc. Munich, Germany).
3
Evaluation of D-pinitol's Effects on Diabetic Cells
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D-pinitol (purity: 95%, Lot No: 441252) and streptozotocin (STZ) were from Sigma Chemicals Co. (St. Louis, MO, USA). Paraformaldehyde (PFA), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), trypsin/EDTA solution, D-glucose, and 3-(4,5-dimethylthiazol)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Solarbio (Beijing, China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from GIBCO (Grand Island, NE, USA). Hoechst 33258 and PI were purchased from Beyotime (Shanghai, China). AGE-BSA was purchased from Abcam (Cambridge, UK). ELISA kits and necrosulfonamide (NSA) were purchased from R&D systems (Minneapolis, MN, USA). Annexin V/PI Kit was from Key Gen (Nanjing, China). The primers were synthesized by Sangon Biotechnology (Shanghai, China). The MFG-E8 antibody was purchased from MBL (Woburn, MA, USA) and R&D (Minneapolis, MN, USA), and antibodies RIPK1, MLKL, phospho-MLKL, and β-action were purchased from Abcam (Cambridge, UK). RIPK3 and HMGB1 antibodies were purchased from Proteintech (Wuhan, China). All other reagents are standard commercial high purity materials.
4
Western Blot Analysis of Protein Expression
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Western blot analysis was carried out as reported previously[15 (link)]. Total protein was extracted from brain tissues of experimental rats using RIPA Lysis Buffer (MultiSciences, China). The BCA Protein Quantification Kit (MultiSciences, China) was used to detect protein concentration. The proteins were separated by SDS-PAGE gel (MultiSciences, China). Primary antibodies (RIPK1, RIPK3, p-RIPK3, MLKL, p-MLKL, GAPDH) were purchased from Abcam (United States). ChemDocTM XRS+ System (Bio-Rad, United States) was used to detect the protein bands. GAPDH was the internal reference protein.
5
Immunohistochemical Analysis of RIPK1 and VEGF-C
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Five micrometer paraffin-embedded sections were stained for RIPK1 (Abcam, Cambridge, UK, 1:500), vascular endothelial growth factor-C (VEGF-C) (Santa Cruz, New York, USA, 1:350) and LYVE-1 (R&D Systems, Minneapolis, USA, 1:250) using MaxVisionTM HRP-Polymer IHC Kit according to the manufacturer’s instructions (Maixin, Fuzhou, People’s Republic of China). Five high-power fields (400×) were randomly selected from each slice. RIPK1 and VEGF-C protein expression in each slice was semiquantitatively analyzed by calculating the mean optical density (MOD) using the Image-Pro plus 6.0 software (Media Cybernetics, Maryland, USA). Expression intensity of RIPK1 was categorized as high or low based on the MOD medians.
6
Quantitative Western Blot Analysis
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After washing with ice-cold PBS, cells were lysed on ice in 100μL RIPA buffer containing 100mM PMSF. Cell lysates were collected and centrifuged at 14,000 rpm for 10 mins at 4°C. The protein lysates were then mixed with 5×loading buffer and denatured by boiling for 5 mins at 100°C, separated on 10% polyacrylamide gels (Invitrogen), and transferred to PVDF (polyvinylidene fluoride) membranes. Membranes were blocked with 5% skim milk in PBS containing 0.1% Tween 20 (PBS-T) for 2.5 hrs. Thereafter, membranes were incubated overnight in antibodies targeting RIPK1 (Abcam, Cambridge, UK, 1:1000), AP-1 (Abcam, Cambridge, UK, 1:1000), P-AP-1 (Abcam, Cambridge, UK, 1:1000), JNK (Abcam, Cambridge, UK, 1:1000), P38MAPK (Abcam, Cambridge, UK, 1:1000), ERK1/2 (Abcam, Cambridge, UK, 1:1000), E-cadherin (Abcam, Cambridge, UK, 1:500) N-Cadherin (Abcam, Cambridge, UK, 1:1000) and VEGF-C (Santa Cruz, New York, USA, 1:1000) at 4°C. After conjugating with respective HRP-coupled secondary antibodies, the protein–antibody complexes were detected using chemiluminescence (Life Technologies, USA) and recorded on Hyperfine-ECI detection film. Target protein expression was semi-quantified relative to GAPDH (loading control) expression.
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