The primary antibodies used for immunocytochemistry included rabbit anti-zonula occludin (ZO)-1 (617300; Invitrogen, Carlsbad, CA, USA), mouse anti-ZO-1 (339100; Invitrogen), mouse anti-cellular retinaldehyde-binding protein (CRALBP; ab15051; Abcam, Cambridge, MA, USA), rabbit anti-paired box 6 (Pax-6; PRB-278P; BioLegend, San Diego, CA, USA), and mouse anti-Bestrophin (BEST)1 (ab2182; Abcam). The secondary antibodies used were goat anti-mouse and goat anti-rabbit IgG conjugated to Alexa Fluor 488, 594, or 647 (Life Technologies). For Western blotting, the antibodies used included rabbit anti-PGC1 alpha (NBP1-04676; Novus Biologicals, Littleton, CO, USA), rabbit anti-b-Actin (CST 4967S; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-PTEN (CST 9552S; Cell Signaling Technology), rabbit anti-SIRT1 (CST 9475; Cell Signaling Technology), rabbit anti-SIRT3 (ab217319; Abcam), rabbit anti-TFAM (CST 8076S; Cell Signaling Technology), mouse anti-TOMM20 (ab56783; Abcam), and mouse anti-VDAC (ab14734; Abcam). The secondary antibodies used were goat anti-mouse IgG and goat anti-rabbit IgG (H+L; Southern Biotech, Birmingham, AL, USA). The details of the antibodies used are listed in Supplementary Table S2 .
Nbp1 04676
Manufactured by Novus Biologicals
Sourced in United States
NBP1-04676 is a laboratory product offered by Novus Biologicals. It is a tool used for scientific research purposes. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg h l, by Southern Biotech (2 mentions)
Goat anti mouse igg, by Southern Biotech (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Ab15051, by Abcam (1 mentions)
Ab56783, by Abcam (1 mentions)
Ab14734, by Abcam (1 mentions)
Ab2182, by Abcam (1 mentions)
Ab217319, by Abcam (1 mentions)
Pax6 prb 278p, by BioLegend (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Cst 9475, by Cell Signaling Technology (1 mentions)
Magicmark xp western protein standard, by Thermo Fisher Scientific (1 mentions)
Western blot hrp substrate series, by Takara Bio (1 mentions)
Lncrna fish kit, by RiboBio (1 mentions)
Fish probe, by RiboBio (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Ab58610, by Abcam (1 mentions)
Bnip3, by Cell Signaling Technology (1 mentions)
Sc 376672, by Santa Cruz Biotechnology (1 mentions)
11 protocols using nbp1 04676
1
Immunocytochemistry and Western Blot Antibodies
2
Mitochondrial Protein Expression Analysis
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The procedures followed those previously described.1 The primary antibodies purchased were as follows: β−actin (#4967) and mitochondrial transcription factor A (#8076) from Cell Signaling Technology; TOMM20 (ab56783), COXIV (ab202554), and voltage-dependent anion channel (ab235143) from Abcam; and peroxisome proliferator-activated receptor-gamma coactivator 1-α (NBP1-04676) from Novus Biologicals. Goat anti-mouse IgG and goat anti-rabbit IgG (H + L; Southern Biotech) were used as secondary antibodies. MagicMark XP Western Protein Standard (Thermo Fisher Scientific) was used as the molecular weight marker. The protein bands were visualized using a Western BLoT HRP substrate series (TakaraBio).
3
Co-localization of LINC00842 and PGC-1α in PDAC
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Co-localization of LINC00842 and PGC-1α protein was analyzed by RNA FISH performed with a lncRNA FISH Kit (RiboBio) and immunofluorescence staining of PGC-1α antibody. Briefly, PDAC cells were fixed and permeabilized in PBS containing 0.5% of Triton X-100. Hybridization with the FISH probes designed by RiboBio was carried out overnight in a humidified chamber at 37 °C in dark. 4′,6-diamidino-2-phenylindole and Cy3 channels were used to detect the signals. Immunofluorescence staining of PGC-1α was performed using PGC-1α antibody (1:200, NBP1-04676, Novus Biologicals, verification of antibody specificity was shown in Supplementary Fig. 15a ) and Alexa Fluor® 488 conjugated secondary antibody (1:500, A-21206, Invitrogen). Cell nuclei were counterstained with DAPI. Images were obtained with Olympus FV1000 confocal microscope.
4
Quantification of Mitochondrial Proteins
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Snap-frozen quadriceps muscle tissues were homogenized in lysis buffer as reported (19 (link)). Cytosolic and mitochondrial proteins were separated. After Western blotting of the samples, protein bands were detected and analyzed using a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA). Voltage-dependent anion channel protein (VDAC) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a house keeping gene with relatively stable expression) were used as the loading control for mitochondrial protein and cytosolic protein, respectively. Results were expressed as the integrated optical density (IOD) relative to VDAC or GAPDH. VDAC (1:1000, #4661) and BNIP3 (1:1000, #12396) antibodies were purchased from Cell Signaling Technologies (Danvers, MA). Antibodies for LC3-I/II (1:1000, ab58610), ubiquitin-binding protein p62 (1:1000, ab91526), and cytochrome C (Cyt-C, 1:1000, ab13575) were from Abcam (Cambridge, United Kingdom). ATP5B antibody (1:1000, ARP48185_T100) was from Aviva Systems Biology (San Diego, CA). Mitochondrial transcription factor A (TFAM) (1:100, sc-376672) and nuclear respiratory factor-1 (NRF-1) (1:100, sc-33771) antibodies were from Santa Cruz Biotechnology (CA). PGC-1α antibody (1:2000, NBP1-04676) was from Novus Biological (CO). GAPDH antibody (1:1000, 60004-1-Ig) was from Proteintech (Chicago, IL).
5
Mitochondrial Biogenesis and Dynamics Proteins
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To study the expression of mitochondrial biogenesis and dynamics proteins, we prepared whole cell lysates with a buffer containing 10 mM Tris, 10 mM EDTA, 100 mM NaCl, 0.5% Triton, 10% glycerol, and protease inhibitors. Culture lysates were mixed with 4X loading buffer and denatured at 95°C for 10 min. Then, samples were subjected to SDS-PAGE in 10% acrylamide gels at 100 V for 100 min and transferred to nitrocellulose membranes at 250 mA for 120 min. Membranes were blocked with 5% non-fat milk in TBS 1X-Tween for 1 h. The primary antibodies SIRT1 (mouse, 1:1,000, Novus Biologicals, 1F3), PGC-1α (rabbit, 1:500, Novus Biologicals, NBP1-04676), DRP1 (rabbit, 1:2,000, Novus Biologicals, NB110-55288), Mfn1 (rabbit, 1:1,000, NBP1-51841), and β-actin (mouse, 1:1000, Santa Cruz Biotechnology, AC-15) were incubated overnight. Anti-rabbit-HRP (1:5,000, Santa Cruz, Biotechnology, sc-2004) and anti-mouse-HRP (1:5,000 Santa Cruz, Biotechnology, sc-2005) were used as secondary antibodies and were incubated for 1 h. Immunoreactive signals were detected using the Western Lighting kit (Perkin Elmer, USA) andquantified with an Odyssey imaging system (Li-Cor, Lincoln, NE, USA).
6
SH-SY5Y Protein Extraction and Western Blot
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SH‐SY5Y cells were harvested with trypsin and lysed on ice in 1% (v/v) Triton X‐100 in PBS supplemented with protease and phosphatase inhibitors or 0.1% (v/v) sodium dodecyl sulphate (SDS), 10 mM Tris pH 7.4, 150 mM NaCl supplemented with DNase and protease/phosphatase inhibitors. Protein lysates (20 μg) were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to Hybond P (GE Healthcare, Little Chalfont, UK). Blots were probed with antibodies against Nrf2 (ab31163; Abcam, Cambridge, UK), p62 (610833; BD Biosciences, Oxford, UK), prohibitin 1 (2426; Cell Signaling Technology, Beverly, MA, USA), cytochrome oxidase (COX) subunit IV (ab14744; Abcam), LC3B (2775; Cell Signaling Technology), cathepsin D (ab6313; Abcam), TOM20 (FL‐145; Santa Cruz Biotechnology, Santa Cruz, CA, USA), TFEB (ab2636; Abcam), DDK (clone 4C5; Origene), lamin A (ab26300; Abcam), PINK1 (BC100‐494; Novus Biologicals, Cambridge, UK), PGC‐1α (NBP1‐04676; Novus Biologicals) or β‐actin (ab82618; Abcam). Bands were detected with respective horse radish peroxidase‐linked secondary antibodies (Dako, Carpinteria, CA, USA) and enhanced chemiluminescence (Pierce). Density of bands was determined using ImageJ software (NIH, Bethesda, MD, USA) or Image Lab software 5.1 (Bio‐Rad Laboratories, Hercules, CA, USA).
7
Quantifying NAD+/NADH and PGC1α Acetylation
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The NAD+ and NADH contents were measured using a NAD+/NADH Assay Kit (Beyotime) according to the manufacturer's recommendation. The amounts of PGC1α acetylation were performed as previously described (Woldt et al, 2013 ). Briefly, cells were transfected with plasmid of Flag‐PGC1α expression vector, and where indicated, mGPDH siRNA or plasmid was used to knock down or overexpress mGPDH as indicated. The cells were harvested, and the PGC1α acetylation was determined by immunoprecipitation of lysates with anti‐PGC1α antibody (Novus NBP1‐04676, 2 μg per sample), followed by Western blot analysis using antibodies to acetylated lysine (1:500, Santa Cruz, sc‐32268). The input was blotted with antibody to Flag‐epitope tag (1:1,000, Cell Signaling Technology, 8146s).
8
Western Blot Analysis of Mitochondrial Proteins
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A Western blot was carried out to detect the expression of carnitine palmityl transferase 1 (CPT1α, ab234111, Abcam, UK), pyruvate kinase isozymes M2 (PKM2, 4053S-100UL, Cell Signaling Technology, CST, USA), 3-hydroxy-3-methylglutaryl- Coenzyme A synthase 2 (HMGCS2, ab137043, Abcam, UK), Na+-coupled monocarboxylate transporter (SMCT1, orb101289, Biorbyt, UK), succinyl-CoA:3-ketoacid CoA transferase (SCOT, ab224250, Abcam, UK), peroxisome proliferator activated receptor co-activator-1α (PGC1α, NBP1-04676, Novus, USA) (PGC1α, ab191838, Abcam, UK), mitochondrial fusion protein 1 (Mfn1, 14739S, Cell Signaling Technology, CST, USA), mitochondrial dynamin-related protein 1 (Drp1, NB110-55288, Novus, USA), Bcl-2 (A19693, Abclonal, Wuhan, China), Bax (A12009, Abclonal, Wuhan, China), and silent information regulation 2 homolog 3 (SIRT3, 2627S, Cell Signaling Technology, CST, USA) proteins. Among them, CPT1α, PKM2, HMGCS2, SMCT1, SCOT, PGC1α, Mfn1, Drp1, Bcl2, Bax, and SIRT3 were all rabbit polyclonal antibodies with a dilution of 1:1000, while GAPDH was a mouse monoclonal antibody with a dilution of 1:3000.
9
Protein Expression Analysis in Neuron Cultures
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DNs were lysed with sample buffer (62.5 mM Tris–HCl buffer [pH 6.8] containing 2% sodium dodecyl sulfate [SDS], 5% β‐mercaptoethanol, and 10% glycerol) and incubated at 95°C for 5 min. The proteins in the cell lysates were electrophoresed using SDS‐polyacrylamide gel electrophoresis, and immunoblotting was performed using rabbit anti‐nuclear receptor‐related 1 (NURR1; #10975‐2‐AP; Proteintech), mouse anti‐TH (#66334‐1‐Ig; Proteintech), rabbit anti‐forkhead box protein A2 (FOXA2; #22474‐1‐AP; Proteintech), rabbit anti‐peroxisome proliferator‐activated receptor gamma coactivator 1 alpha (PGC‐1α; #NBP1‐04676; Novus Biologicals), mouse anti‐postsynaptic density protein 95 (PSD‐95; #MAB1598; Millipore), rabbit anti‐paired‐like homeodomain 3 (PITX3; #CSB‐PA010844LA01HU; CUSABIO), mouse anti‐microtubule‐associated protein 2 (MAP2; #M9942; Sigma–Aldrich), rabbit anti‐delta like non‐canonical Notch ligand 1 (DLK1; #10636‐1‐AP; Proteintech), rabbit anti‐DAT1; #22524‐1‐AP; Proteintech) and mouse anti‐α‐tubulin (#sc‐32,293; Santa Cruz Biotechnology) antibodies. The immunoreactive bands were detected using ECL Prime (Cytiva) and analyzed using LAS‐1000 pro (Fuji Film) and Image Gauge software (Fuji Film). α‐tubulin was used as an internal control. To normalize the protein expression, the chemiluminescent signal was divided by the chemiluminescent signal of α‐tubulin.
10
Antibody-based detection of iron-related proteins
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Antibodies targeting the following proteins were used: TFRC (ab214039, Abcam, Cambridge, UK), FPN (NBP1-21502, Novus Biologicals, Englewood, CO, USA), FTH1 (ab65080, Abcam), SLC7A11 (600-401-GU3, Rockland Immunochemicals, Pottstown, PA, USA), GPX4 (ab125066, Abcam), GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA), PGC1α (NBP1-04676, Novus Biologicals), SHARPIN (ABF128, Millipore, Burlington, MA, USA), β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA), VDAC1/3 (ab14734, Abcam), PRMT5 (sc-376937, Santa Cruz Biotechnology), SDMA (SYM10; 07-412, Millipore), SOX10 (sc-365692, Santa Cruz Biotechnology), MITF (ab12039, Abcam), LC3B (NB100-2220, Novus Biologicals), NRF2 (#12721, Cell Signaling Technology), Parkin (#4211, Cell Signaling Technology), and BNIP3L/NIX (#12396, Cell Signaling Technology). For NRF2 detection, samples were pretreated with 50 μM MG132 (FUJIFILM Wako Pure Chemical Corporation, San Diego, CA, USA) for 3 h. Complex I/III/V was detected using Total OXPHOS Human WB Antibody Cocktail (ab110411, Abcam). Erastin, EPZ015666, ATN-161, and RGD peptide were purchased from Selleck Chemicals (Houston, TX, USA). TGF-β was purchased from BioLegend (San Diego, CA, USA). RSL3, SC-514, and ferrostatin-1 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Deferoxamine was obtained from Cayman Chemicals (Ann Arbor, MI, USA).
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