Cell lysate or in vivo protein samples were separated by 10% SDS-PAGE, then electrophoretically transferred to 0.45 μm poly-vinylidene difluoride membranes (Millipore). Membranes were blocked for 1 h in Blocking Reagent (TOYOBO, Osaka, Japan), probed with primary antibodies followed by labeling with HRP-coupled secondary antibodies (Amersham, Piscataway, NJ), and then visualized using chemiluminescence reagent (ECL; Amersham). Images were captured using ImageQuant LAS 4000 (GE healthcare Bio-Sciences, Marlborough, MA, USA). Quantitative densitometric analyses were conducted using ImageQuant TL (GE healthcare Bio-Sciences). The protocol was slightly modified for detection of HDAC6 in cortex. Briefly, cerebral cortex was homogenized in RIPA buffer supplemented with 1 μM microstatin, 1 μM MG115, 40 μM Leupeptin, 100 μM ABSF, and 2 mM sodium orthovanadate, separated by 7.5–15% gradient SDS-PAGE and electrophoretically transferred to 0.45 μm poly-vinylidene difluoride membranes (Millipore). Membranes were blocked for 1 h with ECL Prime Blocking Reagent (Amersham, Piscataway, NJ) prior to immunoblotting, visualization, and densitometry as described above.
Ecl prime blocking reagent
Manufactured by Cytiva
Sourced in Japan
ECL Prime Blocking Reagent is a laboratory reagent designed to reduce non-specific binding in Western blotting procedures. It is a solution of proteins and other components that can effectively block unoccupied binding sites on membranes, preventing the attachment of primary and secondary antibodies to areas where the target protein is not present.
Automatically generated - may contain errors
Lab products found in correlation
Blocking reagent, by Toyobo (1 mentions)
0.45 μm polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Trans blot turbo mini pvdf transfer pack, by Bio-Rad (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Ubiquitin fk2, by Enzo Life Sciences (1 mentions)
Cathepsin d, by Enzo Life Sciences (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica camera (1 mentions)
Fusion fx device, by Vilber (1 mentions)
Extra page one precast gel 5 20, by Nacalai Tesque (1 mentions)
Mouse anti β actin monoclonal antibody ac 15, by Merck Group (1 mentions)
Rabbit anti gfp polyclonal antibody fl, by Santa Cruz Biotechnology (1 mentions)
Chemi lumi one ultra, by Nacalai Tesque (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
6 protocols using ecl prime blocking reagent
1
Western Blot Protein Detection Protocol
2
TetR Protein Expression Verification
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The expression of TetR after genomic integration was confirmed with western blotting. For SDS-PAGE, Any kDMini-PROTEAN® TGX Stain-Free™ precast gels were used. The protein amount was adjusted to 40 μg protein per sample, mixed with 4x protein loading buffer and heated to 95°C for 5–10 min. The SDS-PAGE gels were run with 140 V in 1× Laemmli buffer.
For western blotting, the Trans-Blot® Turbo™ Transfer system was utilized according to the manufacturer's protocol. Total protein was blotted to a PVDF membrane using the Trans-Blot® Turbo™ Mini PVDF transfer pack (0.2 μm PVDF, Bio-Rad). After blotting the proteins to the membrane, the membrane was blocked with 2% (w/v) ECL Prime blocking reagent (Amersham) in 1× TBST for 1.5 h. The anti-TetR antibody (C. Berens, FAU Erlangen) was used in a 1:5000 dilution, the anti-β-actin (Sigma-Aldrich) in a 1:7000 dilution. Detection was performed with the Clarity™ Western ECL Blotting substrate (Bio-Rad).
For western blotting, the Trans-Blot® Turbo™ Transfer system was utilized according to the manufacturer's protocol. Total protein was blotted to a PVDF membrane using the Trans-Blot® Turbo™ Mini PVDF transfer pack (0.2 μm PVDF, Bio-Rad). After blotting the proteins to the membrane, the membrane was blocked with 2% (w/v) ECL Prime blocking reagent (Amersham) in 1× TBST for 1.5 h. The anti-TetR antibody (C. Berens, FAU Erlangen) was used in a 1:5000 dilution, the anti-β-actin (Sigma-Aldrich) in a 1:7000 dilution. Detection was performed with the Clarity™ Western ECL Blotting substrate (Bio-Rad).
3
Recombinant Nanobodies for VacA/VacB
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Recombinant nanobodies with the Fc portion of rabbit IgG, which recognize specifically VacA or VacB were previously characterized (Bosmani et al., 2020) . The other following antibodies were used: pan-vacuolin (Dr. M. Maniak (Jenne et al., 1998) ), VatA (Dr. M. Maniak (Jenne et al., 1998) ), p80 (purchased from the Geneva Antibody Facility), cathepsin D (Dr. J. Garin (Journet et al., 1999) ), ubiquitin FK2 (Enzo Life Sciences), and GFP (pAb from MBL Intl., mAb from Abmart). Goat antimouse or anti-rabbit IgG coupled to AlexaFluor 488, AlexaFluor 594, AlexaFluor 647 (Invitrogen) or to HRP (Brunschwig) were used as secondary antibodies.
After SDS-PAGE separation and transfer onto nitrocellulose membranes (Protran), immunodetection was performed as previously described (Schwarz et al., 2000) but with ECL Prime Blocking Reagent (Amersham Biosciences) instead of non-fat dry milk. Detection was performed with ECL Plus (Amersham Biosciences) using a Fusion Fx device (Vilber Lourmat). Quantification of band intensity was performed with ImageJ.
For immunofluorescence, infected D. discoideum cells were fixed with ultra-cold methanol (MeOH) at the indicated time points and immunostained as previously described (Hagedorn et al., 2006) . Images were recorded with a Leica SP8 confocal microscope using a 63×1.4 NA oil immersion objectives.
After SDS-PAGE separation and transfer onto nitrocellulose membranes (Protran), immunodetection was performed as previously described (Schwarz et al., 2000) but with ECL Prime Blocking Reagent (Amersham Biosciences) instead of non-fat dry milk. Detection was performed with ECL Plus (Amersham Biosciences) using a Fusion Fx device (Vilber Lourmat). Quantification of band intensity was performed with ImageJ.
For immunofluorescence, infected D. discoideum cells were fixed with ultra-cold methanol (MeOH) at the indicated time points and immunostained as previously described (Hagedorn et al., 2006) . Images were recorded with a Leica SP8 confocal microscope using a 63×1.4 NA oil immersion objectives.
4
Western Blot Analysis of XRCC4 and PAXX
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Preparation of total cell lysate and western blot analysis were carried out as previously described [31 , 36 (link), 37 (link)] with the following modifications: The total proteins were electrophoresed on Extra PAGE One Precast Gel 5–20% (Nacalai Tesque, Kyoto, Japan, 13064‐64); the molecular weight marker used was a 3‐Color prestained XL‐ladder (APRO Science, Tokushima, Japan, SP‐2140); and the membranes were blocked in Blocking One (Nacalai Tesque, 03953‐95) or ECL Prime Blocking reagent (GE Healthcare Bio‐Sciences. Corp., Piscataway, NJ, USA, RPN418) for 30 min at room temperature. The following antibodies were used: goat anti‐XRCC4 polyclonal antibody (C‐20; Santa Cruz Biotechnology, Santa Cruz, TX, USA, sc‐8285), anti‐C9orf142 (PAXX) polyclonal antibody (Abcam, Cambridge, UK, ab126353), rabbit anti‐GFP polyclonal antibody (FL; Santa Cruz Biotechnology, sc‐8334), and mouse anti‐β‐actin monoclonal antibody (AC‐15; Sigma‐Aldrich, St. Louis, MO, USA, A5441). In accordance with the manufacturer's instructions, protein bands were detected using a Select Western Blotting Detection System (GE Healthcare Bio‐Sciences. Corp., RPN2235) or Chemi‐Lumi One Ultra (Nacalai Tesque, 11644‐40), and visualized using the ChemiDoc XRS System (Bio‐Rad, Hercules, CA, USA).
5
Western Blot Analysis of HeLa Cell Lysates
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HeLa cells were lysed in lysis buffer (50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM EDTA, supplemented with protease inhibitor cocktail (Roche)). Lysates were mixed with 2x sample buffer (62.5 mM Tris-HCl (pH6.8), 2% SDS, 10% Glycerol, 5% 2-mercaptethanol, 0.02% Bromophenol blue), electrophoresed on Laemmli SDS-PAGE gel, and transferred onto PVDF membranes (Immobilon-P, Millipore) using a Bio-Rad Trans-Blot system. Membranes were blocked with 1% ECL prime Blocking Reagent (Cytiva) in TBS (137 mM NaCl, 2.6 mM KCl, 25 mM Tris-HCl (pH7.4)) with 0.1% Triton X-100 for 30 min, followed by incubation with primary antibody in blocking buffer overnight at 4 °C. Horse radish peroxidase-conjugated secondary antibodies were incubated for 60 min at room temperature and developed using ECL prime (Cytiva). Blots were imaged with a LAS 1000 imager (Fujifilm). All Western blots were reproduced at least three times as biological replicates. Source data are provided as a Source Data file.
6
Western Blot Analysis of Protein Markers
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Cells were lysed using the RIPA Lysis Buffer System supplemented with a protease inhibitor cocktail, sodium orthovanadate, and PMSF (Santa Cruz Biotechnology, Dallas, TX). Total protein (20 μg) samples were loaded into each well of SDS-PAGE gel (10%) for separation by electrophoresis and transferred to a PVDF membrane (0.2 μm; GE HealthCare, Chicago, IL). Membranes were blocked for 1 h with TBS-Tween 20 containing 5% ECL prime blocking reagent (Cytiva, Tokyo, Japan) and incubated overnight at 4 °C with primary antibodies against FoxO1 (1:1000; #2880; Cell Signaling Technology), αSMA (1:1000; #ab7817; Abcam), Krt14 (1:1000; #ab7800; Abcam), Krt5 (1:1,000; #ab52635; Abcam), β-actin (1:1000; #A5060; Sigma-Aldrich), Eda (1:500; #PA5-72840; Invitrogen), Eda2r (1:1000; #BS-7111R; Bioss), phospho-NF-κB p65 (1:1000; #3033; Cell signaling), and NF-κB p65 (1:1000; #8242; Cell signaling). After washing with TBS-T, the membranes were incubated with secondary antibodies (1:5000) against anti-rabbit IgG (#NA934; Cytiva) and anti-mouse IgG (#NA391; Cytiva). Immunoreactivity was detected using ECL Prime western blotting detection reagent (Cytiva) and photographed using an Amersham Imager 600 (Cytiva).
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