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Rabbit anti lc3

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Rabbit anti-LC3 is a primary antibody that specifically recognizes the Microtubule-associated proteins 1A/1B light chain 3B (LC3B) protein. LC3B is a marker for autophagosomes and is widely used to monitor autophagy.

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Lab products found in correlation

Rabbit anti actin, by Merck Group (4 mentions) Rabbit anti beclin 1, by Cell Signaling Technology (4 mentions) Rabbit anti p62, by Cell Signaling Technology (4 mentions) Rabbit anti caspase 3, by Cell Signaling Technology (3 mentions) Rabbit anti atg7, by Cell Signaling Technology (3 mentions) Triton x 100, by Merck Group (3 mentions) Mouse anti tubulin, by Merck Group (2 mentions) Rabbit anti cleaved caspase 3, by Cell Signaling Technology (2 mentions) Rapamycin, by Merck Group (2 mentions) Rabbit anti fip200, by Proteintech (2 mentions) Phosphatase inhibitor cocktail, by Merck Group (2 mentions) Bis tris gel, by Thermo Fisher Scientific (2 mentions) P62 sqstm1, by Novus Biologicals (2 mentions) Mouse anti lamp1, by BD (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Bca protein assay kit, by Beyotime (2 mentions) Beclin 1, by Cell Signaling Technology (2 mentions) Mouse anti β actin, by Cell Signaling Technology (2 mentions) Complete ultra tablet, by Roche (2 mentions) Rabbit anti α tubulin, by Cell Signaling Technology (2 mentions)

46 protocols using rabbit anti lc3

1

Immunofluorescent Analysis of LC3 and LAMP1

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Slides containing 5 μm tissue sections were deparaffinized followed by antigen unmasking in sodium citrate buffer. Then, the tissue sections were incubated with 1% bovine serum albumin (room temperature for 30 min) to block non-specific antibody binding. Following this, the samples were incubated at 4°C overnight with the following primary antibodies: rabbit anti-LC3 (1:100 dilution) (12741-Cell Signaling Technology, Danvers, Massachusetts, USA) and mouse anti-LAMP1 (1:100 dilution) (15665- Cell Signaling Technology, Danvers, Massachusetts, USA). Then, after washing with 0.1M PBS (pH 7.4), samples were incubated with Alexa Fluor-488-conjugated goat anti-rabbit IgG (1:100 dilution; catalog no. RBaf48801; Fcmacs, Nanjing, China) and Tritc-conjugated goat anti-mouse IgG (1:100 dilution; catalog no. BA1089; Boster, Wuhan, China), secondary antibodies for two hours at 37°C. Nuclei were counterstained with 4′,6-diamidino-2- phenylindole (DAPI) (catalog no. 13G04A76; Boster, Wuhan, China). Visualization of fluorescence on sections were observed under an Olympus BX53 microscope and fluorescent images were captured with an Olympus DP73 digital color camera
Tarique I., Shi Y., Gandahi N.S., Ding B., Yang P., Chen C., Vistro W.A, & Chen Q. (2020). in vivo cellular evidence of autophagic associated spermiophagy within the principal cells during sperm storage in epididymis of the turtle. Aging (Albany NY), 12(10), 8987-8999.
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2

Western Blot Analysis of Key Proteins

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For Western blot analysis cells were lysed in RIPA buffer and different amounts of total proteins were separated by electrophoresis through SDS-PAGE gels and blotted into nitrocellulose membranes. Mouse anti-RTN-1C (Abcam, ab8961) was diluted 1:1000 rabbit anti-LC3 (Cell Signaling, 3868 S) was diluted 1:1000, rabbit anti-syntaxin 17 (Abcam, ab116113) was diluted 1:200, rabbit anti-HA (Sigma, H6908) was diluted 1:2000. Rabbit anti-actin (Sigma, A2066) diluted 1:2000, mouse anti-GADPH diluted 1:1000 (Santa Cruz, sc-47724) and mouse anti-tubulin 1:2000 (Sigma, T-4026) were used as loading controls. Secondary antibodies (Biorad, 1721011, 1706515) were diluted 1:5000.
D’ Eletto M., Risuglia A., Oliverio S., Mehdawy B., Nardacci R., Bordi M, & Di Sano F. (2019). Modulation of autophagy by RTN-1C: role in autophagosome biogenesis. Cell Death & Disease, 10(12), 868.
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3

Hypoxia Signaling Pathway Analysis

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CoCl2 was purchased from Sigma-Aldrich. Caspase inhibitor z-VAD-FMK was obtained from BD Pharmingen. The following primary antibodies were used: rabbit anti-Hif1α (SCBT), rabbit anti-Ambra1 (SDI), rabbit anti-Actin (Sigma), rabbit anti-LC3 (Cell Signaling), rabbit anti-cleaved Caspase 3 (Cell Signaling), rabbit anti-p62 (MBL), rabbit anti-PARP. Goat anti-mouse and goat anti-rabbit HRP-conjugated secondary antibodies (Sigma) were used for western blotting. Alexa Fluor 488 and 555 conjugated secondary antibodies (Invitrogen) were used for immunofluorescence analysis.
Pourpirali S., Valacca C., Merlo P., Rizza S., D’Amico S, & Cecconi F. (2015). Prolonged Pseudohypoxia Targets Ambra1 mRNA to P-Bodies for Translational Repression. PLoS ONE, 10(6), e0129750.
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4

Antibody Reagent Identification for Cell Analysis

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The rabbit anti-Bcl-XL, the rabbit anti-caspase-3, the rabbit anti-LC3, the mouse anti-PARP1, the rabbit anti-actin antibodies were from Cell Signaling (ref. no. 2764, 9662, 2775, 9546, 4970 respectively). The rabbit anti-ATG6, the rabbit anti-total Bax and the rabbit anti-total Bak were from Santa Cruz Biotechnology (ref. no. sc-11427, sc-493 and sc-832 respectively). The mouse anti-caspase-1 was from Adipogen (ref. no. AG-20B-0048). The rabbit anti-ATG5 was from Abcam (ref. no. ab108327). The rat anti-MLKL was from Merck Millipore (ref. no. MABC604). The mouse FITC-labelled anti-CD19 was from Beckman Coulter (ref. no. A07768).
Heulot M., Chevalier N., Puyal J., Margue C., Michel S., Kreis S., Kulms D., Barras D., Nahimana A, & Widmann C. (2016). The TAT-RasGAP317-326 anti-cancer peptide can kill in a caspase-, apoptosis-, and necroptosis-independent manner. Oncotarget, 7(39), 64342-64359.
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5

Crocetin Modulates TGF-β Signaling

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Crocetin was obtained from MP Biomedicals, CA, United States. Crocetin was purified by high-performance liquid chromatography (HPLC) with 98% purity and then dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) with a 0.1% final concentration in the culture medium. TGF-β was purchased from Merck Millipore (Burlington, MA, United States). Antibodies including rabbit anti-Caspase3 (#2723), mouse anti-Bcl-2 (#15071), mouse anti-Bax (#89477), rabbit anti-TGF-β (#3711), rabbit anti-α-SMA (#19245), rabbit anti-p-AMPKα (#50081), rabbit anti-p-Beclin-1 (#35955), mouse anti-SIRT-1 (#8469), rabbit anti-LC3 (#12741), and rabbit anti-NDRG2 (#5667) were purchased from Cell Signaling Technology (CST) Inc., Beverly, MA, United States. Mouse anti-GAPDH (sc-365062) and mouse anti-p-mTOR (sc-293113) were obtained from Santa Cruz Biotechnology, Inc., CA, United States.
Guo H., Ruan C., Zhan X., Pan H., Luo Y, & Gao K. (2022). Crocetin Protected Human Hepatocyte LO2 Cell From TGF-β-Induced Oxygen Stress and Apoptosis but Promoted Proliferation and Autophagy via AMPK/m-TOR Pathway. Frontiers in Public Health, 10, 909125.
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6

Characterization of Muscle Protein Expression

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Gastrocnemius muscle was homogenized in RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, and protease inhibitors. Protein was quantified using the Bio-Rad kit (Sigma-Aldrich, Poole, UK) and transferred to nitrocellulose membrane after electrophoresis using 8–15% polyacrylamide gels. Bands were scanned and quantified using Gene Tool system (SynGene – Frederick, MD, USA). Primary antibodies used include rabbit anti-SERCA1 (Cell Signaling technology; #12293, Danvers, MA, USA), rabbit anti-SERCA2 (Cell Signaling technology; #4388, Danvers, MA, USA) and rabbit anti-LC3 (Cell Signaling technology; #2775, Danvers, MA, USA) at 1:1000 dilution each. Secondary antibody used was HRP-linked anti-rabbit IgG (Cell Signaling technology; #7074, Danvers, MA, USA) at 1:10,000 dilution. All image intensities were normalized to protein intensity based on the ponceau stain.
Qaisar R., Bhaskaran S., Ranjit R., Sataranatarajan K., Premkumar P., Huseman K, & Van Remmen H. (2018). Restoration of SERCA ATPase prevents oxidative stress-related muscle atrophy and weakness. Redox Biology, 20, 68-74.
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7

Immunohistochemical Analysis of SMAD4, LC3, and γ-H2AX

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IHC staining was performed as previously described(29 (link)). Briefly, tumor sections were deparaffinized, rehydrated, and treated with 10 mM citrate buffer (pH 6.0) at 95°C to retrieve antigens. After quenching endogenous peroxidase activity with H2O2 and blocking with 10% horse serum, the sections were incubated sequentially with the primary antibodies mouse anti-SMAD4 (1:100; Cell Signaling Technology Inc. CA, USA), rabbit anti-LC3 (1:100; Cell Signaling Technology Inc. CA, USA), rabbit anti-γ-H2AX (1:100; Cell Signaling Technology Inc. CA, USA), biotinylated secondary antibodies, and the Avidin-Biotin Complexes (ABC) reagent (Gene Tech. San Francisco, CA, USA). The immunostaining was visualized with 3.3-diaminobenzidine (Gene Tech. San Francisco, CA, USA). The sections were then counterstained with hematoxylin. Negative controls were performed in each case by replacing the primary antibody with PBS.
Wang F., Xia X., Yang C., Shen J., Mai J., Kim H.C., Kirui D., Kang Y., Fleming J.B., Koay E.J., Mitra S., Ferrari M, & Shen H. (2018). SMAD4 gene mutation renders pancreatic cancer resistance to radiotherapy through promotion of autophagy. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(13), 3176-3185.
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8

Copper-Compound Modulation of Cell Death

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Chemicals and antibodies were purchased as follows: H2DCF (Beyotime, S0033), N-acety-l-cysteine (Sigma, A7250), 3-methyladenine (Sigma, M9281), propidium iodide (Sigma, P4170), chloroquine diphosphate (Sigma, C6628), Necrostatin-1 (Sigma, N9037), rapamycin (Sigma, R0395), SB203580 (Sigma, S8037), SP100625 (Sigma, S5567), Lipofectamine 2000 (Invitrogen, 11668-019), rabbit anti-LC3 (Cell Signaling, 3868S), rabbit anti-ATG7 (Sigma, A2856), rabbit anti-MAPK14/p38α (Bioworld, BS3566), rabbit anti-p-MAPK11/12/13/14 (Santa Cruz, sc-17852R), rabbit anti-p-MAPKAPK2/MK2 (Cell Signaling, 3316P), rabbit anti-PARP1 (Cell Signaling, 9542P), rabbit anti-CASP3 (Cell Signaling, 9662s), rabbit anti-GAPDH (Earthox, E021060), rabbit anti-SQSTM1 (MBL, PM045), mouse anti-ACTB (Bioworld, BSAP500060), goat anti-mouse IgG (Bioworld, BS13278), goat anti-rabbit IgG (Bioworld, BS12478), ANXA5-EGFP Apoptosis Detection Kit (Vigorous, A002), copper (II) chloride (CuCl2, Beijing Shiji, China). HYF127c/Cu was freshly prepared by mixing equal volumes of 100 mmol HYF127c and 100 mmol CuCl2, then diluted to the appropriate concentrations before treatment.
Zhong W., Zhu H., Sheng F., Tian Y., Zhou J., Chen Y., Li S, & Lin J. (2014). Activation of the MAPK11/12/13/14 (p38 MAPK) pathway regulates the transcription of autophagy genes in response to oxidative stress induced by a novel copper complex in HeLa cells. Autophagy, 10(7), 1285-1300.
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9

Immunofluorescence Staining of Neural Markers

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Primary antibodies used were: mouse anti-BrdU (G3G4; DSHB), mouse anti-GFAP (Thermo Fisher Scientific), rhodamine-conjugated mouse anti-BrdU (Millipore), mouse anti-nestin (Rat-401), mouse anti-NeuN (Millipore), mouse anti–Nqo-1 (Santa Cruz Biotechnology), mouse anti-p62 (Abcam), mouse anti ubiquitin (Santa Cruz Biotechnology), mouse antivinculin (Sigma-Aldrich), rabbit anti-Atg5 (Novus Biologicals), rabbit anti-Atg16L1 (Abgent), rabbit anti-GFAP (Dako), rabbit anti-Ki67 (Spring Bioscience), rabbit anti-LC3 (Cell Signaling), rabbit anti-NBR1 (Cell Signaling), rabbit anti-Nrf2 (Abcam), rabbit anti-p62 (Enzo), rabbit anti-Sox2 (Millipore), rabbit anti-TAX1BP1 (Abgent), rabbit anti-Fip200 (ProteinTech), rabbit anti-olig2 (Millipore), rat anti-Ki67 (BioLegend), and guinea pig antidoublecortin (anti-DCX; EMD Millipore). Secondary antibodies were goat anti–rabbit IgG-FITC, goat anti–rabbit IgG–Texas red, goat anti–mouse IgG-FITC, goat anti–mouse IgG–Texas red, goat anti–mouse IgG-HRP, and goat anti–rabbit IgG-HRP (Jackson Immunology).
DHE and EUK-8 were purchased from Sigma-Aldrich. EUK-134 was purchased from Cayman Chemical. DCFDA, MitoTracker Red, and MitoTracker Green were purchased from Invitrogen.
Wang C., Chen S., Yeo S., Karsli-Uzunbas G., White E., Mizushima N., Virgin H.W, & Guan J.L. (2016). Elevated p62/SQSTM1 determines the fate of autophagy-deficient neural stem cells by increasing superoxide. The Journal of Cell Biology, 212(5), 545-560.
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10

Western Blot and Immunohistochemistry for Autophagy

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Western blot analysis and immunohistochemistry have been carried out as described previously33 (link). Antibodies for research were below: rabbit anti-PKM2, rabbit anti-Beclin1, rabbit anti-LC3, rabbit anti-p-AKT, and mouse/rabbit secondary antibody were purchased from Cell Signaling Technology; rabbit anti-AMPKα1, rabbit anti-p-AMPKα1, rabbit anti-SIRT1, and mouse anti-GAPDH were purchased from Santa Cruz Biotechnology. Immunoblotting of LC3 usually gives two bands: LC3-I and LC3-II. The LC3-II/LC3-I ratio correlates with the number of autophagosomes and is applied as the marker for autophagic maker34 (link). For immunohistochemistry analysis, we considered PKM2-positive expression when cases with more than 30% cancer cells were stained.
Li X., Deng S., Liu M., Jin Y., Zhu S., Deng S., Chen J., He C., Qin Q., Wang C, & Zhao G. (2018). The responsively decreased PKM2 facilitates the survival of pancreatic cancer cells in hypoglucose. Cell Death & Disease, 9(2), 133.
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