The following materials were purchased from commercial sources: Rat embryonic ventricular myocardium H9c2 from the Chinese Academy of Sciences Cell Bank (Beijing, China); Dulbeccos modified Eagles medium (DMEM) medium and fetal bovine serum (FBS) from HyClone Laboratories (Logan, UT, USA); GRP78, CHOP, caspase-12, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primary antibody, and HRP secondary antibody from Proteintech Group, Inc. (Wuhan, China); BCA protein concentration assay kit, Annexin V-FITC apoptosis detection kit, and Fluo-3 AM calcium ion detection kit from Beyotime Biotechnology, Inc. (Nantong, China); TRIzol kit, reverse transcription kit, and RT-PCR kit from Invitrogen (Carlsbad, CA, USA); primer synthesis from Takara (Dalian, China); hydrogen peroxide, sevoflurane, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from Sigma (St. Louis, MO, USA).
Caspase 12
Manufactured by Proteintech
Sourced in United States, China
Caspase-12 is a member of the caspase family of proteases. It functions as a key mediator of apoptosis, or programmed cell death, in response to specific cellular stresses. The core function of Caspase-12 is to facilitate the activation of the apoptotic pathway.
Automatically generated - may contain errors
Lab products found in correlation
Grp78, by Proteintech (8 mentions)
Caspase 3, by Proteintech (6 mentions)
Bcl2 associated x (bax), by Proteintech (4 mentions)
β actin, by Proteintech (3 mentions)
Bcl 2, by Proteintech (3 mentions)
Cytochrome c, by Proteintech (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Caspase 9, by Proteintech (2 mentions)
Grp94, by Proteintech (2 mentions)
P erk, by Proteintech (2 mentions)
Sevoflurane, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions)
Bca protein concentration assay kit, by Beyotime (1 mentions)
Tissue or cell total protein extraction kit, by Sangon (1 mentions)
Timp1, by Proteintech (1 mentions)
Goat anti mouse igg antibody, by Proteintech (1 mentions)
12 protocols using caspase 12
1
Apoptosis and Calcium Signaling in H9c2 Cardiomyocytes
2
Protein Expression Analysis in Lung Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from lung tissues or cells using Tissue or Cell Total Protein Extraction Kit (Sangon Biotech). Equivalent protein from different samples was separated by protein electrophoresis, following by transformation onto PVDF membranes (Merck Millipore, Billerica, MA, USA). The membranes were incubated with the anti-rabbit SIRT1, TIMP-1, MMP-9, CHOP, GRP78, caspase-12 or caspase-3 antibodies (1:1000 dilution, Proteintech, Wuhan, China) at 4 °C overnight after immersed into blocking buffer. After the membranes were washed with TBST for several times, goat anti-mouse IgG antibody (1:5000, Proteintech) labelled with horseradish peroxidase were incubated with the membranes as a secondary antibody. Anti-mouse β-actin antibody (1:5000, Proteintech) was used as a reference protein for normalization. The grey levels of the protein bands were examined by Image J software.
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ventricular tissue, cardiomyocytes, or EV pellets were lysed by radioimmunoprecipitation assay buffer (RIPA, Solarbio, R0010) for 30 min on ice along with vortex every 5 min. Total protein concentration of tissues, cells, or EVs was measured in line with the manufacturer’s instructions of BCA protein assay kit. Protein samples were separated by electrophoresis in SDS-PAGE gel with convenient concentrations based on the diverse molecular weight and then transferred into a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Milwaukee, MI, USA). The PVDF membrane was blocked with 5% skim milk followed by primary antibodies incubation overnight at 4 °C. The membrane was then incubated with the secondary antibodies after 3 times wash by TBST (5 min per time). The blots were exposed by adding ECL reagent (Millipore, WBULS0100, Burlington, MA, USA) and then imaged by gel doc XR system (Bio-Rad, Hercules, CA, USA). Antibodies involved in this study were listed as follows: Alix (WL03063, Wanleibio, China), CD63 (ab68418, Abcam, Cambridge, MA, USA), TSG101 (ab125011, Abcam), GRP78 (11,587-1-AP, Proteintech, Wuhan, China), IRE1α (#3294, Cell Signaling Technology, MA, USA), CHOP (15,204-1-AP, Proteintech), Caspase-12 (55,238-1-AP, Proteintech), Tubulin (ab6046, Abcam). The gray value of each blot was quantified by FIJI ImageJ.
4
Dissecting the Unfolded Protein Response Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We bought primary antibodies of GRP78 (Cat#48119), CHOP (SAB 40744) from Signalway Antibody (USA). The primary antibodies of XBP1 (Cat#24868-1-AP), BCL2 (Cat#12789-1-AP), BAX (Cat#50599-2-IG), Caspase3 (Cat#19677-1-AP), Caspase12 (Cat#24868-1-AP), Cytochrome C (Cat# 66264-1-IG), were β-Actin (Cat# 66009-1-IG) were supplied by Proteintech (China). We purchased the primary antibodies from Cell Signaling Technology (USA), and the antibodies include cleaved Caspase3 (Cat#9661s), Caspase3 (Cat#14220S) mTOR (Cat#2983s), p-mTOR ser2448 (Cat#5536s), 4E-BP1 (Cat#9644), p-4E-BP1 (Cat#2855), P70S6K (Cat#9202), and p-P70S6K (Cat#9205). ATF6 (Cat#WL02407) was from Wanleibio (China). We performed the study by using secondary antibodies of HRP-conjugated Goat anti-Rabbit IgG (Cat#HS101-01) and HRP-conjugated Goat anti-mouse IgG (Cat#HS201-01). These antibodies were supplied by TransGEN Biotech (China). Sangon Biotech (China) provided us with Tunicamycin (Cat#A611129-0005).
5
Testis Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from frozen testis tissues using RIPA buffer and protease inhibitors. Proteins were fractionated by 10% SDS-PAGE. The membranes were blocked in TBST solution (5% milk in TBST with 0.05% Tween-20) and incubated with primary antibody overnight at 4 °C. The primary antibodies were used for ɣ-H2AX, caspase-8 (1: 1,000, Cell Signaling Technology, Danvers, MA, USA), caspase-3, caspase-9, caspase-12, Bax, Bcl-2 and β-actin (1:1000, Proteintech). Then, the membrane was incubated with appropriate second antibody (1:10000, Signalway Antibody) and visualized using an enhanced chemiluminescence detection kit (Millipore Corp., Massachusetts, USA).
6
Western Blot Analysis of Oxidative Stress Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung samples were homogenized in lysis buffer. Total protein from each sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membrane (Bio-Rad). The membranes were blocked by TBS-0.05% Tween-20 (TBS-T) with 5% nonfat dry milk for 60 min and were then incubated with mouse anti-rat NRF2 (1 : 500, Proteintech, USA), GCS (1 : 200, Beijing Biosynthesis Biotechnology Co., Ltd.), HO-1 (1 : 200, Beijing Biosynthesis Biotechnology Co., Ltd.), SOD 2 (Santa Cruz Biotechnology, Inc.), GRP 78 (1 : 2000, Proteintech, USA), PERK (1 : 1000, Bioworld, USA), p-PERK (1 : 200, Santa Cruz Biotechnology, Inc.), eIF2α (1 : 1000, Abcam, USA), p- eIF2α (1 : 1000, Cell Signaling, USA), ATF4 (1 : 1000, Protein tech, USA), CHOP (1 : 500, Biolworld, USA), bax (1 : 1000, Proteintech, USA), caspase 12 (1 : 1000, Proteintech, USA), and goat anti-mouse β-actin (1 : 2000, Santa Cruz, California, USA) antibodies in TBS-T with 5% BSA overnight at 4°C, respectively. A corresponding secondary antibody treatment is followed by enhanced chemiluminescence (ECL). The relative protein expression was quantified by densitometry using image quant software (Molecular Dynamics). The result of NRF2, GCS, HO-2, SOD 2, GRP78, p-PERK/PERK, p- eIF2α/eIF2α, ATF4, CHOP, bax, caspase 3, and caspase 12 was represented by the relative yield to the β-actin, respectively.
7
Immunofluorescence Analysis of ER Stress Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence analysis was performed as previously described 30 (link). Briefly, NP cells or tissues attached to slides were fixed with 4% paraformaldehyde, then permeabilized with 0.2% Triton X-100 in PBS. The slides were washed in PBS and blocked with 2% bovine serum albumin (BSA) in PBS for 2 h at 37 °C, and then incubated with primary antibodies against: GRP78 (1:50), GRP94 (1:50), CHOP (1:100), caspase-3 (1:50), or caspase-12 (1:100) (Proteintech). After washed twice, the slides were subsequently treated with secondary goat anti-rabbit antibody (Boster) at 37 °C for 2 h. Nuclei were co-stained for 5 min with 0.1 g/mL DAPI (Beyotime, Nantong, China), and images were captured under a microscope (Olympus, BX53; Melville, NY, USA).
8
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were isolated using a protein extraction kit (Beyotime, Nantong, China) according to standard protocols. The antibodies used were: CD63 (1:1000), TSG101 (1:1000), Alix (1:1000), GRP78 (1:2000), GRP94(1:1000), CHOP (1:1000), caspase-3 (1:500), caspase-12 (1:1000) (Proteintech), p-PERK (1:1000), PERK (1:1000), ATF6 (1:1000), p-IRE1α (1:1000), IRE1α (1:1000) (Abcam), ERK (1:1000), p-ERK (1:1000), AKT(1:1000), p-AKT (1:1000), and GAPDH (1:1000) (Cell Signaling Technology). Horseradish peroxidase (HRP) -conjugated secondary antibodies (Santa Cruz Biotechnology) were used and protein bands were visualized and detected using an enhanced chemiluminescence system. GAPDH was used for normalization. The experiments were performed in triplicate.
9
Antibody-based Analysis of ER Stress Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The information on antibodies is as follows: GAPDH (Proteintech, Chicago, IL, USA; 1:2000), ACSL4 (Abcam, Boston, MA, USA; 1:10,000), GPx4 (Abcam, USA; 1:1000), FTH1(Abcam, USA; 1:1000), GRP78 (Proteintech, Chicago, IL, USA; 1:1000), IRE1 (Proteintech, Chicago, IL, USA; 1:1000), ATF6(Proteintech, Chicago, IL, USA; 1:500), PERK (Proteintech, Chicago, IL, USA; 1:500), P53(Proteintech, Chicago, IL, USA; 1:5000), HO-1(Proteintech, Chicago, IL, USA; 1:500), Caspase12 (Proteintech, Chicago, IL, USA; 1:500), Caspase3 (Proteintech, Chicago, IL, USA; 1:1000), Bax(CST, Danvers, MA, USA; 1:1000). The images were obtained by a chemiluminescence imaging analysis system (ECL from Tanon 5200, Shanghai, China) and the band intensities were analyzed by Tanon Gis analytical software (Shanghai, China).
10
Western Blot Analysis of Apoptosis Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein from cell lysates or tissues lysates were separated by SDS–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and incubated with primary antibodies, including: calpain-9 (1:1000; Proteintech, USA), calpain-8 (1:1000; Abcam, USA), cyclin D1 (1:500; Cell Signaling, USA), cyclin D3 (1:1000; Cell Signaling), CDK4 (1:1000; Cell Signaling), CDK6 (1:1000; Cell Signaling), p21 (1:1000; Cell Signaling), cleaved caspase-3 (1:500; Cell Signaling), cleaved caspase-8 (1:1000; Cell Signaling), cleaved caspase-9 (1:1000; Cell Signaling), and caspase-12 (1:1000; Proteintech), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Santa). Protein expression was visualized by enhanced chemiluminescence assay.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!