Livers from six-week-old WT and Npc1−/− mice were homogenized in RIPA buffer with the complete protease inhibitor cocktail (Thermo Scientific, Waltham, MA USA; # 78430). 12% SDS-PAGE was run and proteins were blotted into Midi nitrocellulose membranes ( Bio-rad, Hercules, CA, USA; #17001915). The primary antibodies used were: anti-NPC1 serum kindly donated by Dr. William Garver (University of Arizona, Tucson, AZ, USA), anti-CTSB (Cell Signaling Technology, Inc., Danvers, MA, USA; (D1C7Y) # 31718), anti- LAMP1 (Cell Signaling Technology, Inc., Danvers, MA, USA; (D2D11) # 9091), anti-StarD3 (Invitrogen, Waltham, MA, USA; PA1-562), and anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA; (6C5) # sc32233). Anti-rabbit IgG HRP-linked Antibody (Cell Signaling Technology, Inc., Danvers, MA, USA; # 7074) and Goat anti-mouse IgG (H+L) (Invitrogen, Waltham, MA, USA; #62-6520) secondary antibodies HRP conjugate were used to visualize proteins in the BOX Model Chemi XRQ Syngene (UK).
Anti ctsb
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-CTSB is a laboratory reagent that detects the presence and measures the levels of the CTSB (Cathepsin B) protein in biological samples. CTSB is a lysosomal cysteine protease involved in various cellular processes. Anti-CTSB can be used in techniques such as Western blotting, immunohistochemistry, and ELISA to study the expression and distribution of CTSB in different cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti lamp1, by Cell Signaling Technology (1 mentions)
Midi nitrocellulose membranes, by Bio-Rad (1 mentions)
Anti p62 sqstm1, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Taurine, by Merck Group (1 mentions)
Cadmium chloride, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Beyotime (1 mentions)
Lyso tracker red, by Beyotime (1 mentions)
Anti beclin 1, by Cell Signaling Technology (1 mentions)
Anti lamp2, by Santa Cruz Biotechnology (1 mentions)
Anti snap29, by Abcam (1 mentions)
Cy3 labeled goat anti rat igg, by Beyotime (1 mentions)
Anti lamp2, by Merck Group (1 mentions)
Anti lc3b, by Merck Group (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
4 protocols using anti ctsb
1
Western Blot Analysis of Npc1-/- Mouse Livers
2
Autophagy Regulation in Cadmium Toxicity
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Cadmium chloride, taurine, anti-LAMP2, anti-LC3B, and anti-p62/SQSTM1 were purchased from Sigma-Aldrich (202,908, St. Louis, MO, USA). DMEM and FBS were obtained from Gibco (Grand Island, NY, USA). Alexa Fluor 488-labeled goat anti-rabbit IgG, Cy3-labeled goat anti-rat IgG, Lyso-Tracker Red (LTR), and Hanks’ Balanced Salt Solution were obtained from Beyotime (Shanghai, China); Anti-β-actin, anti-Atg7, anti-Beclin-1, anti-CTSB, and anti-rabbit IgG were obtained from Cell Signaling Technology Inc (Danvers, MA, USA). anti-LAMP2 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DQ-BSA-Green was purchased from Invitrogen (Carlsbad, CA, USA). Anti-STX17, anti-SNAP29, and anti-VAMP8 were obtained from Abcam (UK). All of the other materials were of analytical grade.
3
Protein Expression Analysis Protocol
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Equal amounts of protein lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Filters were probed with the following specific primary antibodies: anti-CTSB (Cell signaling, Danvers, MA, USA), MMP-9 (Abcam, Cambridge, MA, USA), β-actin (Sigma, St Louis, MO, USA), PI3K and phosphorylated-PI3K (Cell signaling, Danvers, MA, USA), Akt and phosphorylated-Akt (Cell signaling, Danvers, MA, USA), mTOR, and phosphorylated-mTOR (Cell signaling, Danvers, MA, USA). Blots were then incubated with horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL, USA) and visualized by chemiluminescence. The band density was quantified by densitometry using Scion Image software, and normalized to β-actin levels.
4
Western Blot Analysis of Autophagy and Apoptosis
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After treatment, cells were harvested at various time intervals and digested in RIPA buffer with presence of Protease Inhibitor Cocktail (Pierce, Rockford, IL, USA), and protein concentration was quantified using the BCA Protein Assay Kit (Pierce). After separated by SDS/PAGE, proteins were transferred to PVDF membrane (Bio‐Rad, Hercules, CA, USA). Blots were probed with anti‐LC3, anti‐p62/SQSTM‐1, anti‐CTSB, anti‐CTSD, anti‐Bcl‐2, anti‐Bax, anti‐cleaved caspase‐3 (all from Cell Signaling Technology), anti‐LAMP‐1, anti‐β‐tubulin, and anti‐GAPDH (Abcam). The goat anti‐rabbit or goat anti‐mouse horseradish peroxidase‐conjugated IgG was used as secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). Bands were observed by enhanced chemiluminescence (Pierce). Band density was measured by densitometry, quantified using the public domain nih image software (open source imagej software available at http://rsb.info.nih.gov/ij/ ) and normalized to an indicated sample in the identical membrane.
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