Pieces of RV and LV tissue were lysed in RIPA buffer and then flash‐frozen. Lysates were electrophoresed on SDS‐PAGE gels, transferred to a nitrocellulose membrane, and blocked with 5% milk in TBS with 0.05% TWEEN®‐20 (TBST). Blots were incubated overnight with anti‐CD45 antibody (Abcam, 10558) or anti‐α‐tubulin (Abcam, MS‐581‐P1ABX), then washed with TBST and incubated with anti‐rabbit HRP (GE Healthcare, NA9340V, for CD45) or anti‐mouse HRP (GE Healthcare, NXA931V, for α‐tubulin) for 1 hr at room temperature. Blots were washed again and developed with ECL substrate (Thermo Fisher Scientific), and imaged on a digital imager.
Nxa931v
Manufactured by GE Healthcare
The NXA931V is a piece of laboratory equipment manufactured by GE Healthcare. It is a specialized device designed for specific laboratory functions. However, without further details about its core functionality, I am unable to provide a detailed and unbiased description while maintaining the requested level of conciseness and objectivity. Therefore, a comprehensive description is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Na9340v, by GE Healthcare (1 mentions)
Na934v, by GE Healthcare (1 mentions)
Mab1501, by Cell Signaling Technology (1 mentions)
Ubiquitin clone p4d1, by Cell Signaling Technology (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Na934v, by Thermo Fisher Scientific (1 mentions)
P s65 ub, by Cell Signaling Technology (1 mentions)
Atp5a, by Abcam (1 mentions)
Gapdh, by GeneTex (1 mentions)
Ab72149, by Abcam (1 mentions)
Ab32386, by Abcam (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Ab7800, by Abcam (1 mentions)
Precellys system, by Avantor (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Imagequant las 4000 chemiluminescence imager, by GE Healthcare (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
7 protocols using nxa931v
1
Western Blot Analysis of CD45 and α-Tubulin
2
Antibodies for Immunoblotting Analysis
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The following mouse antibodies were used for immunoblotting (WB) in this study: ATP5A (Abcam, ab14748, 1:10,000), actin (Millipore, MAB1501, 1:5,000), Ubiquitin (clone P4D1, Cell Signalling Technology, 3936, 1:1,000), Ubiquitin (clone FK2, MBL, D058-3, 1:1,000). The following rabbit antibodies were used in this study: pS65-Ub (Cell Signalling Technologies, 62802S, 1:1,000), Marf ([16 (link)], 1:1,000), GABARAP/Atg8a (Abcam ab109364, 1:1,000), ref(2)P/p62 (Abcam ab178440, 1:1,000). The following secondary antibodies were used: sheep anti-mouse (HRP-conjugated, GE Healthcare, NXA931V, 1:10,000), donkey anti-rabbit (HRP-conjugated, GE Healthcare, NA934V, 1:10,000).
3
Antibody Immunoblotting and Immunofluorescence
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The following mouse antibodies were used for immunoblotting (WB) and/or immunofluorescence (IF) in this study: ATP5A (Abcam, ab14748, 1:10,000 (WB), 1:300 (IF, adult muscle)), Ubiquitin (clone FK2, MBL, D058‐3, 1:2,000 (WB), 1:250 (IF, adult muscle)), Actin (Millipore, MAB1501, 1:1,000 (WB)), GAPDH (GeneTex, GTX627408, 1:1,000 (WB)). The following rabbit antibodies were used in this study: pS65‐Ub (Cell Signalling Technologies, 62802S, 1:750 (WB), 1:200 (IF, larval muscle), 1:250 (IF, adult muscle)), COXIV and SDHA (both kind gifts from Edward Owusu‐Ansah (Murari et al, 2020 (link)), both 1:2,000 (WB)), Porin (Calbiochem, PC548, 1:5,000 (WB)). The following secondary antibodies were used: sheep anti‐mouse (HRP‐conjugated, GE Healthcare, NXA931V, 1:10,000 (WB)), donkey anti‐rabbit (HRP‐conjugated, GE Healthcare, NA934V, 1:10,000 (WB)), goat anti‐mouse (AlexaFluor 488, Invitrogen, A11001, 1:200 (IF)), goat anti‐rabbit (AlexaFluor 594, Invitrogen, A11012, 1:200 (IF)).
4
Ubiquitination Assay for APC/C Substrates
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Ubiquitination assays were performed with 15 nM recombinant human APC/C, 30 nM UBA1, 150 nM UbcH10, 20 μM ubiquitin, 0.2 μM substrates, 5 mM ATP, 0.25 mg/ml BSA and 25 nM Cdc20 in a 15 μl reaction volume with 40 mM Hepes pH 8.0, 150 mM NaCl, 10 mM MgCl2 and 0.6 mM DTT.
Reaction mixtures were incubated at room temperature for 30 min and terminated by adding SDS/PAGE loading dye. Reactions were analysed by 4–12% NuPAGE Bis‐Tris gels followed by Western blotting with an antibody against the His‐tag of ubiquitin to detect the His‐tag of the ubiquitin‐modified substrates (Clontech, mouse monoclonal, 631212) and HRP‐conjugated sheep anti‐mouse antibody (GE Healthcare, NXA931V).
Detection of Nek2A was performed with the mouse monoclonal antibody (BD Transduction Laboratories, 610594). Detection of cyclin A2 was performed with the rabbit monoclonal cyclin A2 antibody (Abcam, ab32386), detection of securin with the rabbit monoclonal securin antibody (Invitrogen, 700791), detection of Apc4 with the rabbit monoclonal Apc4 antibody (Abcam, ab72149), detection of Apc2 with the rabbit monoclonal Apc2 antibody (Cell Signaling, 12301) and HRP‐conjugated donkey anti‐rabbit antibody (Thermo Fischer, SA1‐200). Primary antibodies were used at a dilution of 1:1,000 and secondary antibodies at a dilution of 1:5,000.
Reaction mixtures were incubated at room temperature for 30 min and terminated by adding SDS/PAGE loading dye. Reactions were analysed by 4–12% NuPAGE Bis‐Tris gels followed by Western blotting with an antibody against the His‐tag of ubiquitin to detect the His‐tag of the ubiquitin‐modified substrates (Clontech, mouse monoclonal, 631212) and HRP‐conjugated sheep anti‐mouse antibody (GE Healthcare, NXA931V).
Detection of Nek2A was performed with the mouse monoclonal antibody (BD Transduction Laboratories, 610594). Detection of cyclin A2 was performed with the rabbit monoclonal cyclin A2 antibody (Abcam, ab32386), detection of securin with the rabbit monoclonal securin antibody (Invitrogen, 700791), detection of Apc4 with the rabbit monoclonal Apc4 antibody (Abcam, ab72149), detection of Apc2 with the rabbit monoclonal Apc2 antibody (Cell Signaling, 12301) and HRP‐conjugated donkey anti‐rabbit antibody (Thermo Fischer, SA1‐200). Primary antibodies were used at a dilution of 1:1,000 and secondary antibodies at a dilution of 1:5,000.
5
Cornea and Epidermis Protein Analysis
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Proteins from cornea and epidermis samples were homogenized with the Precellys system (VWR International, Radnor, PA) in Laemmli extraction buffer containing 2% SDS. Protein concentrations were determined with the Micro BCA protein assay kit (Thermo Fisher Scientific). Thirty µg of total protein per lane were electrophoresed through an ExcelGel SDS 8–18% polyacrylamide gradient gel (GE Healthcare Life Sciences, Chicago) and blotted onto a nitrocellulose membrane. The membrane was incubated with guinea pig anti-K24 (dilution 1:1000) overnight at 4 °C. After washing, the membrane was incubated for 1 h at room temperature with goat anti-guinea pig IgG (Abnova, AB10611, 1:10000) coupled to horseradish peroxidase. Bands were visualized using the enhanced chemiluminescence system (SuperSignal West Dura Extended Duration Substrate, Thermo Fisher Scientific). The membrane was reincubated with monoclonal mouse anti-K14 (Abcam, ab7800, 0.33 µg/ml) and sheep anti-mouse IgG (GE Healthcare, NXA931V, 1:10000) coupled to horseradish peroxidase as a secondary antibody.
6
Auxin-Induced Depletion Kinetics of GRH-1-AID-3xFLAG
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To examine the kinetics of GRH‐1‐AID‐3xFLAG depletion by auxin, synchronized grh‐1(xe135); eft‐3p::luc; eft‐3p::TIR1 L1‐stage animals (HW2434) were cultured in liquid (S‐Basal supplemented with OP50, OD600 = 3, 1,000 animals/ml) at 20°C. After 21 h, when animals had reached early L2 stage, the culture was sampled, split in two, and supplemented with 250 μM auxin or an equivalent amount of vehicle, respectively, followed by hourly sampling. At each time point, 10,000 animals were collected and washed three times with M9 buffer (42 mM Na2HPO4, 22 mM KH2PO4, 86 mM NaCl, 1 mM MgSO4). Lysates were made by disruption (FastPrep‐24, MP Biomedicals, 5 cycles, 25 s on, 90 s off), sonication (Biorupter, Diagnode, 10 cycles, 30 s on, 60 s off) and subsequent boiling. Proteins were separated by SDS‐PAGE and transferred to a PVDF membrane by semi‐dry blotting. Antibodies were used at the following dilutions: mouse anti‐FLAG‐HRP (1:1,000, RRID:AB_439702, Sigma Aldrich Cat #A8592), mouse anti‐Actin, clone C4 (1:5,000, RRID:AB_2223041, Millipore MAB1501), mouse IgG HRP linked (1:7,500, NXA931V, GE Healthcare). We used ECL Western Blotting detection reagent (RPN2232 and RPN2209, GE Healthcare) and ImageQuant LAS 4000 chemiluminescence imager (GE Healthcare) for detection.
7
Western Blot Analysis of Polyglutamine Proteins
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20-50 µg of the RIPA soluble fraction protein samples were supplemented with 1x Laemmli buffer and boiled 95 °C, 5 min. Samples were subjected to SDS-PAGE (100 V for 90 min). Resolved proteins were transferred onto nitrocellulose membrane (225 mA per gel for 60 min) by semi-dry blotting. The membranes were then blocked with skimmed milk (5% in TBS-T for 60 min) followed by incubation with the primary antibody at 4°C overnight (in 5% skimmed milk in TBS-T). The primary antibodies used were mouse anti-Polyglutamine, Milipore MAB1574; mouse anti-Huntingtin polyQ, Developmental Hybridoma Bank, MW1 or mouse anti-GFP; Roche 11814460001. All antibodies were applied in a 1:1000 dilution. Membranes were washed three times for 10 min in TBS-T and incubated with the secondary horse radish peroxidase (HRP) coupled antibody (GE Healthcare, NXA931V; in a 1:3000 dilution) for two hours under agitation at room temperature. Subsequently, the membranes were washed three times in TBS-T for 10 min, and the chemiluminescence signal was detected using the Super Signal® West Femto Maximum Sensitivity Substrate (Thermo Scientific) detection kit according to manufacturer's instructions. Chemiluminescence signals were visualized using the Alliance UVltec system (Biometra) and processed using Image J software.
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