ab75973, by Abcam - Product details

1

Investigating Autophagy and Ferritin in Lung Cells

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Firstly, MLE-12 cells with stable YAP1 overexpression were transfected with LC3-GFP (Beyotime, D2815, China) and cultured in eight-well plates for 24 h. Secondly, the cells in each group were treated with LPS and bafilomycin A1 (BafA1) (Abcam, ab120497, UK) for 24 h. Thirdly, the cells were permeated by 0.03% Triton X-100 (Beyotime, China) for 60 min and then fixed and blocked with 0.1% BSA (Beyotime, China) for 1 h. Finally, the primary anti-ferritin (Abcam, ab75973, UK) and secondary antibodies were applied to the medium in turn. MLE-12 cells were analyzed via confocal laser scanning microscopy and evaluated by fluorescence microscopy (Nikon TE-2000, Tokyo, Japan).
For immunofluorescence staining in lung tissues, the samples were fixed in 4% paraformaldehyde for 1 h, washed completely with PBST (1× PBS added with 0.1% Triton X-100) for three times, and permeabilized with 0.1% Triton X-100 for 1 h. Then, the samples were incubated with primary antibodies including anti-LC3 (1:100, AF5402, Affinity Biosciences) and anti-ferritin (1:100, Abcam, ab75973) for 10 h at 4°C. After three times of washing with PBST, the samples were incubated with secondary antibodies, and nuclei were stained with DAPI. After washing again, the samples were imaged with a confocal microscope (Nikon TE-2000).

Zhang J., Zheng Y., Wang Y., Wang J., Sang A., Song X, & Li X. (2022). YAP1 alleviates sepsis-induced acute lung injury via inhibiting ferritinophagy-mediated ferroptosis. Frontiers in Immunology, 13, 884362.

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2

Cellular ROS Assay and Ferroptosis Markers

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DCFDA/H2DCFDA-Cellular ROS Assay Kit was provided by Abcam (ab113851, Cambridge, MA, USA). Erastin (HY-15763, ferroptosis inducer) was purchased from MedChemExpress (Shanghai, China). The following primary antibodies were used in this study: anti-ATF-4 antibody (ab184909, Abcam, USA), anti-CHAC1 antibody (MA5-26311, Invitrogen, Carlsbad, CA, USA), anti-GPX4 antibody (ab125066, Abcam, USA), anti-transferrin receptor antibody (ab277635, Abcam, USA), anti-ferritin antibody (ab75973, Abcam, USA), anti-Hsp70 antibody (ab2787), and anti-GAPDH antibody (ab8245, Abcam, USA). Goat anti-mouse IgG (HRP) (ab6789, Abcam, USA) and goat anti-rat IgG (HRP) (ab97057, Abcam, USA) were used as secondary antibody for Western blotting.

Xu Y., Zhang N., Chen C., Xu X., Luo A., Yan Y., Lu Y., Liu J., Ou X., Tan Y., Liang Y., Chen L., Song X, & Liu X. (2022). Sevoflurane Induces Ferroptosis of Glioma Cells Through Activating the ATF4-CHAC1 Pathway. Frontiers in Oncology, 12, 859621.

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3

Western Blot Analysis of Cellular Proteins

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Cells were plated at 300,000 cells per well in 6-well plates and grown to confluence. Media was then removed, cells were washed twice with PBS, and 200 μL of RIPA lysis buffer containing sigma protease inhibitor cocktail (P8340, 1:100) was added to each well. Cells were incubated in lysis buffer for 10 min at RT then scrapped and collected. Lysates were analyzed by BCA and normalized for total protein content with lysis buffer then denatured in Laemmli loading buffer with 0.01 M DTT at 100 °C for 10 min. 10 μg of protein from each sample was then loaded onto Biorad Mini-PROTEAN® TGX^™ 4–15% gels and run @ 150V for 55 min then transferred to Immobilon PVDF membrane at 35 V for 1 h. The membrane was blocked with 5% non-fat milk in TBS-T for 1 h at rt then incubated with the indicated antibodies in 2.5% non-fat milk overnight at 4 °C. The blot was then washed three-times with TBS-T and once with PBS-T before incubating with Licor IRDye secondary antibodies in Odyssey blocking buffer (#927-40000) + 0.2% Tween-20 and 0.01% Sodium dodecyl sulfate (SDS). The blot was washed 3 times with PBS-T for 5 min and once with PBS then imaged for fluorescent signal on an Odessey Classic Infrared Imaging System. Ferritin antibodies were obtained from Abcam (Ab75973) and tubulin antibodies were obtained from Sigma (T6199).

Spangler B., Morgan C.W., Fontaine S.D., Vander Wal M.N., Chang C.J., Wells J.A, & Renslo A.R. (2016). A reactivity-based probe of the intracellular labile ferrous iron pool. Nature chemical biology, 12(9), 680-685.

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4

Western Blot Analysis of Cellular Proteins

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Cells were plated at 300,000 cells per well in 6-well plates and grown to confluence. Media was then removed, cells were washed twice with PBS, and 200 μL of RIPA lysis buffer containing sigma protease inhibitor cocktail (P8340, 1:100) was added to each well. Cells were incubated in lysis buffer for 10 min at RT then scrapped and collected. Lysates were analyzed by BCA and normalized for total protein content with lysis buffer then denatured in Laemmli loading buffer with 0.01 M DTT at 100 °C for 10 min. 10 μg of protein from each sample was then loaded onto Biorad Mini-PROTEAN® TGX^™ 4–15% gels and run @ 150V for 55 min then transferred to Immobilon PVDF membrane at 35 V for 1 h. The membrane was blocked with 5% non-fat milk in TBS-T for 1 h at rt then incubated with the indicated antibodies in 2.5% non-fat milk overnight at 4 °C. The blot was then washed three-times with TBS-T and once with PBS-T before incubating with Licor IRDye secondary antibodies in Odyssey blocking buffer (#927-40000) + 0.2% Tween-20 and 0.01% Sodium dodecyl sulfate (SDS). The blot was washed 3 times with PBS-T for 5 min and once with PBS then imaged for fluorescent signal on an Odessey Classic Infrared Imaging System. Ferritin antibodies were obtained from Abcam (Ab75973) and tubulin antibodies were obtained from Sigma (T6199).

Spangler B., Morgan C.W., Fontaine S.D., Vander Wal M.N., Chang C.J., Wells J.A, & Renslo A.R. (2016). A reactivity-based probe of the intracellular labile ferrous iron pool. Nature chemical biology, 12(9), 680-685.

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5

Western Blot Analysis of Antioxidant Proteins

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Liver tissues and L02 cells were homogenized in ice-cold RIPA Lysis Buffer (Beyotime, China) with PMSF. Concentrations of protein samples were detected by BCA protein assay (Beyotime, China). Western blotting was performed as previously described [27 (link)]. Protein samples with equal amounts were separated using SDS/PAGE gels and then transferred to polyvinylidene diﬂuoride (PVDF) membrane. After being blocked with 5% BSA for 1 h, membranes were incubated with specific primary antibodies overnight at 4 °C. The primary antibodies were shown as follows: Anti-GPX4 (Abways, CY6959, 1:1500), Anti-Ferritin (Abcam, ab75973, 1:2000), Anti-SLC7A11 (Abcam, ab175186, 1:2000), Anti-Nrf2 for L02 cells (Abcam, ab62352, 1:1000), Anti-Nrf2 for liver tissues (Abclonal, A11159, 1:1000), Anti-pNrf2 (Abcam, ab76026, 1:2000), Anti-GCLC (Abcam, ab20777, 1:1000), Anti-HO-1 (Abcam, ab189491, 1:1000), Anti-NQO1 (Abcam, 1:2000, ab28947), Anti-GAPDH (Abcam, ab181602, 1:10000). Then the membranes were washed with PBST for 3 times and incubated with Anti-rabbit and Anti-mouse IgG HPR-linked secondary antibodies for 1 h, followed by chemiluminescence detection using ChemiDocTM Touch Imaging System (BIO-RAD, California, USA).

Xu Y., Li Y., Li J, & Chen W. (2022). Ethyl carbamate triggers ferroptosis in liver through inhibiting GSH synthesis and suppressing Nrf2 activation. Redox Biology, 53, 102349.

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6

Western Blot Analysis of Oxidative Stress Markers

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Total protein was isolated by lysing cells with RIPA lysis buffer supplemented with protease inhibitor cocktail. Protein quantification was done with BCA protein assay kit (PC0020, Solarbio, China). Equal quantities of protein samples were then mixed with loading buffer, heated for 10 min at 100 °C, separated on 10% SDS-PAGE gels, and subsequently transferred to PVDF membranes, which were then blocked with 5% nonfat milk for 1 h, exposed overnight (O/N) to specified antibodies at 4 °C, followed by three 15-min TBST rinses, and further exposure to anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (2° Ab, 1:5000) at room temperature (RT) for 1 h, before visualization of the protein bands with ECL reagent and Amersham Imager 600 (General Electric Company, USA). Finally, the protein bands were quantified with Image J gel analysis software. All experiments were repeated three times. Among the primary antibodies (1° Abs) used were: Rabbit monoclonal anti-TERT (ab191523, Abcam, 1:1000); anti-Nrf2 (ab137550, Abcam, 1:1000); anti-SLC7A11 (ab37185, Abcam, 1:1000); anti-Ferritin (ab75973, Abcam, 1:1000); anti-Ferroportin (ab239583, Abcam, 1:1000); anti-GPX4 (ab125066, Abcam, 1:1000); and anti-β-Actin (4970S, Cell Signaling Tech, 1:1000).

Dong H., Xia Y., Jin S., Xue C., Wang Y., Hu R, & Jiang H. (2021). Nrf2 attenuates ferroptosis-mediated IIR-ALI by modulating TERT and SLC7A11. Cell Death & Disease, 12(11), 1027.

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7

Immunohistochemistry Antibody Panel

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Antibodies, catalog numbers, and their sources are listed: LC3B (ab51520), CD68 (ab125212), smooth muscle actin (ab5694), ferritin (ab75973) (Abcam), NCOA4 (SAB1409837) (MilliporeSigma), anti-mouse IgG peroxidase conjugated (W402B, Promega), anti-rabbit IgG peroxidase conjugated (ZE 0614, Vector Laboratories), goat anti-rabbit IgG Alexa Fluor 488 (ab150077) and goat anti-mouse IgG Alexa Fluor 594 (ab150116) (Abcam), and horse anti-mouse biotinylated antibody (BA-2000) and goat anti-rabbit biotinylated antibody (BA-1000) (Vector Laboratories).

Radadiya P.S., Thornton M.M., Puri R.V., Yerrathota S., Dinh-Phan J., Magenheimer B., Subramaniam D., Tran P.V., Zhu H., Bolisetty S., Calvet J.P., Wallace D.P, & Sharma M. (2021). Ciclopirox olamine induces ferritinophagy and reduces cyst burden in polycystic kidney disease. JCI Insight, 6(8), e141299.

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8

Antibody Panel for Protein Analysis

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The following antibodies were used: anti-AKT (ab179463, Abcam, Cambridgeshire, UK), anti-AKT (phospho Ser473, 4060, CST, Boston, United States), anti-P53 (2,524, CST, Boston, United States), anti-P53 (phospho S392, ab33889, Abcam, Cambridgeshire, UK), anti-Ferritin (ab75973, Abcam, Cambridgeshire, UK), anti-GPX4 (ab125066, Abcam, Cambridgeshire, UK), anti-XCT (ab175186, Abcam, Cambridgeshire), anti-α-tubulin (ab7291,Abcam, Cambridgeshire, UK). HRP-conjugated secondary antibodies (ZB-2301 or ZB-2305, ZSGB-BIO, Beijing, China) and fluorochrome conjugated secondary antibody (ab150064, Abcam, Cambridgeshire).

Li S., Lei Z., Yang X., Zhao M., Hou Y., Wang D., Tang S., Li J, & Yu J. (2022). Propofol Protects Myocardium From Ischemia/Reperfusion Injury by Inhibiting Ferroptosis Through the AKT/p53 Signaling Pathway. Frontiers in Pharmacology, 13, 841410.

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9

Muscle Iron Regulation Protein Analysis

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The adductor muscles were homogenized using freeze crusher (SK-100, Tokken Inc., Chiba, Japan) and cell lysis buffer (#9803, Cell Signaling Technology, MA, USA). The protein concentration was determined by DC assay kit (#5000112, Bio-Rad, CA, USA). Thirty micrograms of protein homogenate was separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The expression levels of proteins were detected by an enhanced chemiluminescence kit (#32106, Thermo Scientific, IL, USA). Anti-ferritin (#ab75973, Abcam, UK; dilution 1:1000), anti-TfR1 (#13-6800, Invitrogen, CA, USA; dilution 1:1000), anti-ferroportin (NBP1-21502, Novus Biologicals, CO, USA; dilution 1:1000), anti-tubulin (#PM054-7, MBL, Aichi, Japan; dilution 1:1000), anti-mitochondrial total oxidative phosphorylation (OXPHOS) antibody cocktail including antibodies against C I, C II, C III, and C V (#ab110413, Abcam; dilution 1:1000), anti-NADH dehydrogenase iron-sulfur protein 3 (NDUFS3, #ab110246, Abcam; dilution 1:1000), and anti-GAPDH (#2118, Cell Signaling Technology; dilution 1:1000) antibodies were used.

Okuno K., Naito Y., Yasumura S., Sawada H., Asakura M., Masuyama T, & Ishihara M. (2019). Haploinsufficiency of Transferrin Receptor 1 Impairs Angiogenesis with Reduced Mitochondrial Complex I in Mice with Limb Ischemia. Scientific Reports, 9, 13658.

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10

Immunofluorescence Staining of Cathepsin and Iron Markers

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The injured CAs underwent a series of processes, including excision, fixation, and embedding in paraffin. Then, the CAs were cut into 4 μm thick sections and analyzed through IF staining. Homoplastically, the treated HUVECs were fixed in 4% paraformaldehyde. Next, as previously described [20 (link)], the sections and HUVECs were incubated with primary antibodies against cathepsin B (1 : 100, ab214428, Abcam), cathepsin D (1 : 200, #2284 s, Cell Signaling Technology), ferritin (1 : 50, ab75973, Abcam), and TfR (1 : 50, ab269513, Abcam) at 4°C overnight. After washing three times on the following day, the samples were then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H+L) (1 : 300, A32790, Invitrogen, Carlsbad, CA, USA), Alexa Fluor 555-conjugated donkey anti-rabbit IgG (H+L) (1 : 300, A32794, Invitrogen), Alexa Fluor 488-conjugated goat anti-mouse IgG (1 : 300, A-11001, Invitrogen), and Alexa Fluor 555-conjugated goat anti-mouse (1 : 300, A-21424, Invitrogen) secondary antibodies at 37°C for 1 h. Next, 4,6-diamino-2-phenylindole (SouthernBiotech, Birmingham, AL, USA) was added to each section for coverslipping. Finally, the sections were observed under a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Tokyo, Japan), and ImageJ software was used to quantify the fluorescence intensity.

Wang Y., Bao D., Dong Y., Wei X., Yu J, & Niu C. (2022). α-Lipoic Acid-Plus Ameliorates Endothelial Injury by Inhibiting the Apoptosis Pathway Mediated by Intralysosomal Cathepsins in an In Vivo and In Vitro Endothelial Injury Model. Oxidative Medicine and Cellular Longevity, 2022, 8979904.

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Ab75973

Lab products found in correlation

37 protocols using ab75973

Investigating Autophagy and Ferritin in Lung Cells

Cellular ROS Assay and Ferroptosis Markers

Western Blot Analysis of Cellular Proteins

Western Blot Analysis of Cellular Proteins

Western Blot Analysis of Antioxidant Proteins

Western Blot Analysis of Oxidative Stress Markers

Immunohistochemistry Antibody Panel

Antibody Panel for Protein Analysis

Muscle Iron Regulation Protein Analysis

Immunofluorescence Staining of Cathepsin and Iron Markers

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