Anti lc3 2775

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-LC3 (2775) is a primary antibody product from Cell Signaling Technology. It is designed for the detection of the microtubule-associated protein 1A/1B-light chain 3 (LC3) protein. LC3 is involved in autophagosome formation and is commonly used as a marker for autophagic processes.

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Lab products found in correlation

Staurosporin, by Merck Group (1 mentions) Hrp conjugated anti rabbit 7074, by Cell Signaling Technology (1 mentions) Ag 20b 0014 c100, by Adipogen (1 mentions) Anti p62 5114, by Cell Signaling Technology (1 mentions) Ripa buffer, by Sangon (1 mentions) Gapdh, by OriGene (1 mentions) Anti catalase d4p7b, by Cell Signaling Technology (1 mentions) Anti lamp1 d2d11, by Cell Signaling Technology (1 mentions) Anti gm130 d6b1, by Cell Signaling Technology (1 mentions) Alexa fluor 568 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Anti ha h6908, by Merck Group (1 mentions) Dulbecco s phosphate buffered saline dpbs d8537, by Merck Group (1 mentions) Anti traf6 8028s, by Cell Signaling Technology (1 mentions) Lipofectamine 2000 11668 019, by Thermo Fisher Scientific (1 mentions) Anti ub sc 8017, by Santa Cruz Biotechnology (1 mentions) Anti flag f3165, by Merck Group (1 mentions)

4 protocols using anti lc3 2775

Protease Inhibitors and Antibodies Protocol

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Sodium fluoride (S7920), sodium orthovanadate (220590), phenylmethylsulfonyl fluoride (PMSF) (P7626), aprotinin (A162-B), leupeptin (SP-04-2217-B), Triton X-100 (N150), Earle's Balanced Salt Solution (EBSS) (E3024), thapsigargin (T9033), tunicamycin (T7765), E64 (E3132), melphalan (M2011), and staurosporin (S4400) were purchased from Sigma. MG132 (BML-PI102), Epoxomycin (BML-PI127) or Tlck (BML-PI121), Z-Leu-Leu-Glu-AMC (BML-ZW9345), Suc-Leu-Leu-Val-Tyr-AMC (BML-P802) and Ac-Arg-Leu-Arg-AMC (BML-AW9785) were from Enzo Life Sciences. Pepstatin (EI-9-B) was obtained from Euromedex. Anti-Hsp60 (sc-1722), anti-Ubiquitin (sc-8017), anti-Hsp27 (sc-1048) and anti-Hsp90 (sc-69703) were purchased from Santa Cruz Biotechnology. Anti-PSMB5 (PW8140), anti-PSMB6 (PW8145) and anti-PSMB7 (PW8895) were from Enzo life sciences. Anti-PARP (9542), anti-HspB8 (30595), anti-LC3 (2775) and Hrp conjugated anti-rabbit (7074) antibodies were obtained from Cell Signaling Technology. Hrp-conjugated anti-mouse (P0260) and anti-goat (P0449) antibodies were from Dakopatts. Cleaved caspase 3 (9661) was purchased from Ozyme.

Hamouda M.A., Belhacene N., Puissant A., Colosetti P., Robert G., Jacquel A., Mari B., Auberger P, & Luciano F. (2014). The small heat shock protein B8 (HSPB8) confers resistance to bortezomib by promoting autophagic removal of misfolded proteins in multiple myeloma cells. Oncotarget, 5(15), 6252-6266.

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Autophagy and NLRP3 Inflammasome Regulation in THP-1 Cells

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THP-1 cells coincubated with different RBCs were collected. Wild-type, shNC, and low-NLRP3 expression THP-1 cells were triggered with 800 nmol/L of rapamycin to create the autophagy positive control. THP-1 cells were lysed in a radio immunoprecipitation assay (RIPA) buffer (C500005, Sangon Biotech). The expression of autophagy markers on THP-1 was detected using immunoblotting with anti-p62 (5114) and anti-LC3 (2775, Cell Signaling Technology). Anti-NLRP3 (AG-20B-0014-C100, AdipoGen) and antigasdermin D (GSDMD) (HA601046, Hangzhou HuaAn Biotechnology Co., Ltd.) were applied to determine the activation of NLRP3 inflammasome. GAPDH (TA802519, OriGene Technologies) was used as internal control. Each blot was duplicated three times. BandScan software was utilized after development to analyze and record the gray value of the strip. Relative amounts of p62/GAPDH, LC3II/GAPDH, and LC3II/LC3I were adopted to estimate the autophagy situation of THP-1 cells, while those of NLRP3/GAPDH and GSDMD/GAPDH were used to evaluate the activation of NLRP3 inflammasome.

Li Q., Zhao F., Zhang J., Yang Y., Guo Z., Wang C., Yang Q., Sun Y, & Zhu Z. (2022). NLRP3 Inflammasome Simultaneously Involved in Autophagy and Phagocytosis of THP-1 Cells to Clear Aged Erythrocytes. Journal of Immunology Research, 2022, 1481154.

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Multimarker Immunofluorescence Assay

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For standard immunofluorescence experiments, cells were grown on glass-bottomed six-well plates and fixed with 4% formaldehyde. Blocking was performed with 3% BSA in PBS with 0.1% Triton X-100. Primary and secondary antibody dilutions were performed in the same solution. We used anti–protein disulfide isomerase (2792; Abcam) to detect ER, anti-LC3 (2775S; Cell Signaling Technology) to detect autophagy, anti-LAMP1 (D2D11; Cell Signaling Technology) to detect lysosomes, anticatalase (D4P7B; Cell Signaling Technology) to detect peroxisomes, and anti-GM130 (D6B1; Cell Signaling Technology) to detect the Golgi apparatus. We used as a secondary antibody Alexa Fluor 568–conjugated secondary antibodies (Life Technologies). Nuclei were stained with 1 µg/ml Hoechst 33342 (Life Technologies) during one of the post–secondary antibody washes.

Becuwe M., Bond L.M., Pinto A.F., Boland S., Mejhert N., Elliott S.D., Cicconet M., Graham M.M., Liu X.N., Ilkayeva O., Saghatelian A., Walther T.C, & Farese RV J.r. (2020). FIT2 is an acyl–coenzyme A diphosphatase crucial for endoplasmic reticulum homeostasis. The Journal of Cell Biology, 219(10), e202006111.

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Antibody Validation and Autophagy Modulation

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Anti-Flag (F3165) and anti-HA (H6908) antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Myc (sc40), anti-GAPDH (sc47724), anti-BECN1 (sc48341), and anti-Ub (sc-8017) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-TRAF6 (8028S) and anti-LC3 (2775S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Mouse IgG H&L (HRP) (ab6728) was purchased from Abcam (Cambridge, MA, USA). Goat anti-Rabbit IgG antibody (HRP) (GTX213110-01) was purchased from GeneTex Inc. (Irvine, CA, USA). Lipopolysaccharide (LPS; L3024), dimethyl sulfoxide (DMSO; 472301), paraformaldehyde (P6148), Triton X-100 (T8787), 3-methyladenine (3-MA; M9281), and Dulbecco’s phosphate-buffered saline (DPBS; D8537) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyinosinic-polycytidylic acid (Poly(I:C), tlrl-pic) was purchased from InvivoGen (San Diego, CA, USA). Anti-β-arrestin 2 antibody (PA1-732) and Lipofectamine 2000 (11668019) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Kim J.Y., Shin J.H., Kim M.J., Kang Y., Lee J.S., Son J., Jeong S.K., Kim D., Kim D.H., Chun E, & Lee K.Y. (2023). β‐arrestin 2 negatively regulates lung cancer progression by inhibiting the TRAF6 signaling axis for NF-κB activation and autophagy induced by TLR3 and TLR4. Cell Death & Disease, 14(7), 422.

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