Total cell extracts were prepared as previously described by41 (link). Plasma membrane protein fractions were obtained using the ProteoExtract Native Membrane protein Extraction Kit (Merck Millipore, France) accordingly do the manufacturer’s protocol. Protein samples were resolved by SDS-PAGE and immunoblotted with antibodies to AnxA1, AnxA2, AnxA6 (0.2 μg/ml each, Santa-Cruz Biotechnology, Germany), AnxA5 (2 μg/ml, Sigma-Aldrich, France), α-Catenin (0.5 μg/ml, Life Technologies, France), β-Catenin (0.25 μg/ml, Life Technologies, France), E-Cadherin (0.25 μg/ml, Life Technologies, France), ezrin (0.5 μg/ml, Life Technologies, France), actin (0.8 μg/ml, Sigma-Aldrich, France), or GFP (1 μg/ml, Clontech, France). After incubation with appropriate DyLight Fluor-conjugated secondary antibody (680 or 800 conjugate, Life Technologies, France) blots were revealed by using Odyssey infrared fluorescent system (Li-Cor, France). Conversely, blot with cell membrane factions were incubated with reversible protein stain (Thermo scientific, France).
Anxa6
Manufactured by Santa Cruz Biotechnology
Sourced in Germany, Japan, United States
ANXA6 is a protein that belongs to the annexin family. Annexins are a group of proteins that can bind to phospholipids in a calcium-dependent manner. The core function of ANXA6 is to facilitate membrane-membrane and membrane-cytoskeleton interactions.
Automatically generated - may contain errors
Lab products found in correlation
Reversible protein stain, by Thermo Fisher Scientific (1 mentions)
Anxa5, by Merck Group (1 mentions)
E cadherin, by Thermo Fisher Scientific (1 mentions)
β catenin, by Thermo Fisher Scientific (1 mentions)
Anxa1, by Santa Cruz Biotechnology (1 mentions)
Odyssey infrared fluorescent system, by LI COR (1 mentions)
Anxa2, by Santa Cruz Biotechnology (1 mentions)
Actin, by Merck Group (1 mentions)
Proteoextract native membrane protein extraction kit, by Merck Group (1 mentions)
Hsp70, by Santa Cruz Biotechnology (1 mentions)
Hrp secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Takara Bio (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Tissue ia software, by Leica Microsystems (1 mentions)
Anxa6, by Abcam (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
P akt s473, by Santa Cruz Biotechnology (1 mentions)
Bapta am, by Thermo Fisher Scientific (1 mentions)
Annexin 5 alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
6 protocols using anxa6
1
Plasma Membrane Protein Extraction and Analysis
2
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RIPA lysis buffer (Beyotime, Shanghai, China) was used to extract the total proteins, and its quality was measured by BCA kit (Beyotime, Shanghai, China). The proteins were separated by 10% SDS-PAGE, and the targeted proteins were transferred onto the PVDF membranes according to the proteins’ molecular weight. Membranes were then blocked with 5% non-fat milk and were incubated with the primary antibodies against LC3B (1:2000, Santa Cruz, United States), p62 (1:2000, Santa Cruz, United States), GAPDH (1:2000, Takara, Japan), ANXA6 (1:2000, Santa Cruz, United States), TSG101 (1:1500, Takara, Japan), Alix (1:1500, Takara, Japan), and HSP70 (1:1000, Santa Cruz, United States) at 4°C overnight. Then, the PVDF membranes were cultivated with the secondary HRP antibodies (ThermoFisher Scientific, United States) for 2 h at 37°C, and the ECL system was used to visualize the protein bands, which were quantified and analyzed by using the Image J software.
3
Quantifying Immunostaining in Xenograft Tumors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin-fixed paraffin-embedded tissue sections (8 μm thickness) prepared from xenograft tumors from the AnxA6 down-regulated BT-A6sh5 or the AnxA6-deficient BT-A6A cells were stained as previously described [3 (link)] using the following primary antibodies: RasGRF2 (Abcam, Cambridge, MA), AnxA6 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); EGFR (Cell Signaling Technology, Danvers, MA). For quantification of the immunostaining, slides were digitally scanned at the Digital Histology Shared Resource at Vanderbilt University Medical Center. The stained tissue areas were digitally demarcated and the staining intensity analyzed by using the Tissue IA software (Leica Microsystems).
4
Characterization of Mitochondrial Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Common laboratory reagents were purchased from Life Technologies (Grand Island, NY, USA), Sigma (St. Louis, MO, USA) and Thermo Scientific (Waltham, MA, USA), unless otherwise mentioned. PE, Ang II, Iso and LMB were from Sigma. Ionomycin, BAPTA-AM, fluorescent conjugates and other microscopy consumables were from Life Technologies. Mitochondria Isolation Kit and Co-IP Kits were from Pierce Biotechnology (Rockford, IL, USA). PInh was from Calbiochem (La Jolla, CA, USA). JC-1 Staining Kit was from Cayman Chemicals (Ann Arbor, MI, USA). DAPI, Hoechst 33342, propidium podide (PI), Annexin V-Alexa Fluor 488, TMRM, MitoTracker red FM, CellLight Mitochondria-RFP and BacMam 2.0 system were from Life Technologies. Primary antibodies were procured from the following sources: Anxa6 (monoclonal antibody) from BD (Lexington, KY, USA); Anxa6 (polyclonal antibody), COX IV (cytochrome c oxidase subunit IV), Akt, p-AktS473 and α-SkA from Santa Cruz Biotechnology (Santa Cruz, CA, USA); α-tubulin, cleaved caspase-3, cleaved caspase-9, Parp1 and cleaved Parp1 from Cell Signaling Technology (Beverly, MA, USA); pro-ANP from Abcam (Cambridge, MA, USA); anti-COX I monoclonal antibody from Invitrogen (Carlsbad, CA, USA); Living Colors anti-fluorescent protein antibody (JL-8) from Clontech (Mountain View, CA, USA) and pan-actin from Chemicon (Temecula, CA, USA).
5
Comprehensive EV Protein Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and isolated sEVs were lysed with radio-immunoprecipitation assay (RIPA, Beyotime Biotechnology, China) supplemented with phenylmethanesulfonyl fluoride (PMSF) (10 μL/mL of lysis buffer, Beyotime Biotechnology, China). Then, the protein concentration was measured by bicinchoninic acid assay (BCA, Beyotime Biotechnology, China). Briefly, the solution was mixed with 5 × SDS-PAGE loading buffer (Beyotime Biotechnology, China) and boiled at 95 °C for 10 min. Proteins were separated with SDS-PAGE and transferred to a 0.45 μm polyvinylidene difluoride (PVDF) membrane. After that, the membranes were blocked with 5% (w/v) non-fat milk for 90 min and incubated with primary antibody followed by secondary antibody. The primary antibodies used for immunoblotting: CD63 (1:1000, ABCAm, ab134045), CD9 (1:1000, ABCAm, ab92726), Alix (1:500, Santacruz, sc-53540), TSG101 (1:500, Santa Cruz Biotechnology, sc7964), LaminA/C (1:1000, Servicebio, GB11407), Actin (1:1000, Invitrogen, MA5-15739), ANXA6 (1:500, Santacruz, sc-271,859), ECM1 (1:1000, ABCAm, ab126629), ITGB1 (1:500, Santacruz, sc-374,429). The second antibodies used for immunoblotting: anti-mouse IgG, HRP-linked antibody (1:2000, CST, 7076), anti-rabbit, IgG, HRP-linked antibody (1:2000, CST, 7074).
6
Western Blot Analysis of Cell and EV Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blots for cell and EV lysates were performed by lysing cells and EV pellets in RIPA buffer with Halt protease inhibitor (Thermo Fisher Scientific), loading 10 μg total protein lysate, and using the following antibodies: ANXA1 (1:1,000, abcam #ab214486), ANXA2 (1:1,000, abcam #ab178677), ANXA6 (1:1,000, Santa Cruz Biotechnology #271859), β-actin (1:1,000, Novus #NB600-501, St. Louis, MO), calnexin (1:1,000, Cell Signaling Technologies #2679S, Danvers, MA), CD29 (1:1,000, BioLegend #303002), CD63 (1:1,000, abcam #ab8219), GAPDH (1:1,000, Santa Cruz Biotechnology #0411), GM130 (1:1,000, abcam #ab52649), histone H3 (1:1,000, abcam #ab176842), TOMM20 (1:1,000, abcam #ab205486). Blots were incubated with antibodies overnight at 4 degrees Celsius and imaged using with an Amersham ImageQuant 800 (Amersham, UK). Blots were stripped and reprobed for ANXA1, ANXA2, and GAPDH antibodies using RestoreTM Western Blot Stripping Buffer (Thermo Fisher Scientific). CD29 was used as a loading control for quantification of EV blots as the total CD29+ EV population was unchanged assessed by single EV microarray. GAPDH was used as a loading control for quantification of SMCs and VICs whole cell lysates.
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!