The following antibodies were purchased: anti-SYCP3 (Abcam, ab97672, IF: 1:100), anti-SYCP3 (Abcam, ab15093, IF: 1:100), anti-SYCP1 (Abcam, ab15090, IF: 1:100), anti-γH2AX (MerckMillipore, 16-202A, IF: 1:800), anti-TNP2 (Santa Cruz, sc-393843, IF: 1:50), anti-PRM2 (Briar Patch Biosciences, Hup 2B, IF: 1:300), anti-H4K8ac (ABclonal, A7258, IF: 1:500), anti-ELFN2 (Novus Biologicals, 90569, WB: 1: 1000), anti-H3 (Abcam, ab1791, WB: 1: 2000), anti-GAPDH (Abcam, ab8245, WB: 1: 10000), anti-TUBLIN (Sigma, T8328, WB: 1: 2000), anti-FLAG (Sigma, F3165, IP: 10 µg, WB: 1:2000). For immunofluorescence studies, the following secondary antibodies were used: anti-rabbit (Jackson ImmunoResearch 488, 711225152, IF: 1:350), anti-rabbit (Jackson ImmunoResearch 594, 711585152, IF: 1:350), anti-mouse (Jackson ImmunoResearch 488, 715545150, IF: 1:350). For Western blot analyses, the following secondary antibodies conjugated to Horse Radish Peroxidase were used: anti-rabbit IgG HRP-linked (ABclonal, AS014, WB: 1:5000), anti-mouse IgG HRP-linked (ABclonal, AS003, WB: 1:5000).
T8328
Manufactured by Merck Group
Sourced in United States, United Kingdom
T8328 is a laboratory equipment product from Merck Group. It is designed for general laboratory use. The core function of T8328 is to provide a reliable and consistent method for performing various laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Ab1791, by Abcam (3 mentions)
β tubulin, by Merck Group (3 mentions)
Adi spa 901, by Enzo Life Sciences (2 mentions)
F3165, by Merck Group (1 mentions)
Ab8245, by Merck Group (1 mentions)
Ab15093, by Abcam (1 mentions)
Ab97672, by Abcam (1 mentions)
Stripping buffer, by Thermo Fisher Scientific (1 mentions)
Ab97545, by Abcam (1 mentions)
Ab98929, by Abcam (1 mentions)
Hybond, by Cytiva (1 mentions)
Beta 3 tubulin, by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
Nestin, by Merck Group (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Mab1574, by Merck Group (1 mentions)
Ab68167, by Abcam (1 mentions)
Ab18061, by Abcam (1 mentions)
Akt and phospho akt ser473, by Cell Signaling Technology (1 mentions)
Darpp32, by Abcam (1 mentions)
22 protocols using t8328
1
Meiotic Proteins Immunofluorescence and Western Blot
2
Quantitative Western Blot Analysis
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Proteins from total cell lysates (10 to 20 μg) were separated by SDS-PAGE, transferred onto PVDF membranes (Hybond 0.2 μm; Amersham GE Life Sciences), and probed using the indicated antibodies. The secondary HRP-conjugated antibody was visualized by chemiluminescence (SeraCare developer solution). For anti-tubulin probing, membranes were first treated with stripping buffer (Thermo Scientific), washed, and reprobed. Polyclonal rabbit antibodies against VPS35 (ab97545; abcam), VPS29 (ab98929; abcam), rabbit monoclonal antibody against VPS26 (ab98929; abcam) or mouse monoclonal antibody against β-tubulin (catalog no. T8328; Sigma) were used according to the manufacturer’s instructions. Quantification of immunoblots was performed using ImageJ.
3
Immunoblotting Markers for Huntington's Disease
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Polyclonal anti-htt 1–17 Ab1(1μg/ml) [42 (link)], mAb 1C2 to polyglutamines (MAB1574, 1:1000; Millipore), mAb 3B5H10 to polyglutamines (P1874, 1:10,000; Sigma), p85 PI 3-kinase (1:500; #4292, Cell Signaling), AKT and phospho (Ser473) AKT (1:500; Cell Signaling); ERK and phospho ERK (1:1000; Cell Signaling); β-tubulin (1:4000; T8328, Sigma), beta3-tubulin (1:2000; Sigma), anti-GFAP (1:2000; Millipore), Rac1 (1:2000; Millipore), nestin (1:500; Millipore), DARPP32 (1:5000; Abcam), Islet1 (1:200; University of Iowa Developmental Studies Hybridoma Bank), GAPDH (1:6000; Millipore), α-actinin-2 (1:250; mAb clone EA-53, Sigma) or α-actinin-2 (1:2000; ab68167; Abcam; [31 (link)], α-actinin (2μg/ml; ab18061; Abcam; may cross react with isoforms 1–4).
4
Fractionation of U87MG Cells
5
Cellular Fractionation for Nuclear and Cytoplasmic Extraction
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Cells were fractionated following a previously published protocol with some modifications61 (link). Briefly, monolayers of cells in 10-cm plates were washed twice with ice-cold PBS, followed by gentle scraping of cells. Cells were resuspended with the ice-cold HLB+N buffer (10 mM Tris-HCl, at pH 7.5, 10 mM NaCl, 2.5 mM MgCl2 and 0.5% NP-40) on ice for 5 min. Lysates were layered over a chilled 10% sucrose cushion made in the ice-cold HLB+NS buffer (10 mM Tris-HCl, at pH 7.5, 10 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40 and 10% sucrose) and centrifuged for 5 min at 4 °C at 420g. After centrifugation, the supernatant was collected and served as the cytoplasmic fraction. The nuclear pellet was then treated with the ice-cold nuclei lysis buffer (10 mM HEPES, at pH 7.6, 300 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1 M Urea, and 1% NP-40) after washing. Fractionation efficiency was validated by Western blot using antibody specific to the marker for each fraction: β-tubulin (Sigma, T8328) for the cytoplasmic fraction and rabbit polyclonal U1–70k (Santa Cruz, sc-390899) for the nucleoplasmic fraction.
6
Immunoblotting of Protein Targets
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Membranes were incubated overnight with primary antibodies in 3% milk in PBST. The antibodies used for immunoblotting included rabbit anti-IKZF3 (ab139408, Abcam), rabbit anti-CDK8 (A302-501A, Bethyl Laboratories), and mouse anti-β-tubulin (T8328, Sigma-Aldrich) at a 1:2,000 dilution and rabbit anti-SHC (ab24787, Abcam), rabbit anti-SEPT9 (sc-899, Santa Cruz Biotechnology), and mouse anti-MAVS (sc-166583, Santa Cruz Biotechnology) at a 1:1,000 dilution. Membranes were then incubated with IRDye700-labeled donkey anti-mouse or anti-rabbit or IRDye800-labeled donkey anti-mouse (LI-COR Biosciences) at a 1:5,000 dilution. Blots were detected using an Odyssey® Infrared Imaging System (LI-COR Biosciences).
7
Immunofluorescence Staining of Cytoskeletal Proteins
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Male and female fixed spindles slides were washed in PBS containing 0.4%Kodak Photo flo (Kodak) for 5 min, followed by 0.1% PBS-Triton X and blocked in 1XPBS-antibody dilution buffer (ADB) before being incubated over night at 4°C with the primary antibody. Primary antibodies used were: rabbit anti-ADD1 (GTX101600, Genetex. Dilution 1:100), rabbit anti-MYO10 (24565-1-AP, Proteintech. Dilution 1:100) and rabbit anti-β-tubulin (T8328, Sigma. Dilution 1:500). After overnight incubation, slides were washed to remove the unbound antibodies and incubate for 2 hours at 37°C with Alexafluor™ secondary antibodies (Molecular Probes Eugene OR, USA). Slides were washed and mounted with Prolong Gold antifade (Molecular Probes). Image acquisition was performed using a Zeiss Imager Z1 microscope under 20X, 40X or 63X magnifying objectives, at room temperature. Images were processed using ZEN 2 (Carl Zeiss).
8
Profiling Protein Expression Changes
9
Mitochondrial Complex Protein Quantification
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The individual mitochondrial complex determination were previously performed, supplied and determined by Ydfors et al. (2016 ). The proteins of individual electron transport chain (ETC) complexes were detected by western blot analysis using a human OXPHOS Cocktail (ab110411, Abcam) containing five monoclonal antibodies specific for complex I subunit NDUFB8, complex II subunit SDHB, complex III subunit UQCRC2, complex IV subunit COX II, and complex V subunit ATP5A. The protein levels were quantified by densitometry for three time points including, before the first HIIT session (T1) and pre‐session 5 (T5) and pre‐session 9 (T9) (Ydfors et al. 2016 ). We calculated the total OXPHOS content for each time point by taking the sum of the protein densities for the five proteins above. The proteins were normalized with the internal control β‐tubulin (T8328, Sigma).
10
Immunofluorescence Staining of Monolayers
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HPAEC postconfluent monolayers were stained as previously described (Kruse et al., 2019 (link)). Briefly, cells were washed with warm HBSS buffer containing both Ca2+ and Mg2+, fixed with 4% formaldehyde or a mixture of 4% formaldehyde and 0.1% glutaraldehyde (for tubulin staining) for 15 min at room temperature, permeabilized with 0.1% Triton X-100 for 15 min, and blocked using 3% BSA for 1 h. The samples were incubated with primary antibodies against the protein of interest (VE-cadherin [anti-goat; sc-6458; Santa Cruz]; cingulin [anti-rabbit; NBP1-89602; Novus Biologicals]; GEF-H1 [anti-mouse; ab90783; Abcam]; tubulin [anti-mouse; T8328; Sigma Aldrich]) or Alexa Fluor 647 phalloidin at 1:100 overnight at 4°C and thereafter with secondary antibodies 1:100 at room temperature for 1 h. Cells were mounted using Fluoromount-G (Southern Biotech).
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