Proteins were resolved by SDS-PAGE, transferred to nitrocellulose filters, revealed with ECL and detected using a Fusion FX7 imager (Vilber-Lourmat, France). SNAP i.d. protein detection system (Millipore) was used for Flag (ubiquitin) and β-Tubuline immunoblots. The following antibodies were used: mouse monoclonal anti-Myc 9E10 (produced in the laboratory), mouse monoclonal anti-Mdm2 (MABE331, Millipore), mouse monoclonal anti-β-Tubulin (T4026, Sigma), rabbit polyclonal anti-NUB1 (BML-PW9685, Enzo), mouse monoclonal anti-Flag (M2, Sigma-Aldrich).
Fusion fx7 imager
Manufactured by Vilber
Sourced in France
The Fusion FX7 imager is a laboratory equipment designed for the visualization and analysis of various biological samples. It utilizes a high-resolution camera and specialized software to capture and process images of gels, blots, and other samples. The core function of the Fusion FX7 is to provide researchers and scientists with a versatile tool for the documentation and quantification of their experimental results.
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Lab products found in correlation
Anti β actin, by Merck Group (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Snap i d protein detection system, by Merck Group (1 mentions)
T4026, by Merck Group (1 mentions)
Anti chop, by Cell Signaling Technology (1 mentions)
Anti phospho eif2α, by Cell Signaling Technology (1 mentions)
Hpr conjugated secondary antibody, by Boster Bio (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Sc 6243, by Novus Biologicals (1 mentions)
Mouse anti myc tag, by Cell Signaling Technology (1 mentions)
Amersham hybond p 0.45 pvdf blotting membrane, by Cytiva (1 mentions)
Ecl prime western blotting system, by Cytiva (1 mentions)
Ecl select reagent, by GE Healthcare (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Anti cbp antibody, by Merck Group (1 mentions)
Anti flag monoclonal antibody, by Merck Group (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
15 protocols using fusion fx7 imager
1
Protein Immunodetection Workflow
2
Protein Detection Using Vilber Imager
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Experiments were performed as described in Simonetti et al.21 except that detection was done with a Vilber Lourmat Fusion Fx7 imager.
3
Immunoblot Analysis of UPR Signaling
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Proteins were resolved by SDS-PAGE, transferred to nitrocellulose filters, revealed with ECL and detected using a Fusion FX7 imager (Vilber-Lourmat, France). The following antibodies were used: rabbit monoclonal anti-PERK (C33E10, CTS), rabbit polyclonal anti-BIP (C50B12, CTS), rabbit polyclonal anti-IRE1α (14C10, CTS), rabbit monoclonal anti-ATF6 (D4Z8V, CST), rabbit polyclonal anti-human NUPR1 antibody (home made) and anti-β-actin (A5316, Sigma).
4
Western Blot Analysis of UPR Markers
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Proteins were resolved by SDS-PAGE, and transferred to nitrocellulose membranes for 1 h. Then, membranes were blocked 1 h at room temperature with TBS (tris-buffered saline), 5% BSA, and blotted overnight in TBS 5% BSA containing primary antibodies at 1:500 overnight with corresponding antibodies at 4 °C. Subsequently, the blot was washed and incubated with HPR-conjugated secondary antibody (Boster, Pleasanton CA, USA) for 1 h at room temperature at 1:5000 before being revealed with ECL (enhanced chemo-luminescence). The acquisition was performed with a Fusion FX7 imager (Vilber-Lourmat, Sud Torcy, France). The following primary antibodies were used: rabbit polyclonal anti-PARP (#9542, Cell Signalling, Danvers MA, USA), mouse polyclonal anti-CHOP (#2895, Cell Signalling), rabbit polyclonal anti-Phospho-IRE1α (#PA1-16927, Thermofisher Scientific), rabbit polyclonal anti-Phospho-eIF2α (#3398, Cell Signalling), and mouse monoclonal β-actin (#A5316, Sigma).
5
Western Blot Analysis of Protein Targets
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Proteins were resolved by SDS-PAGE, and transferred to nitrocellulose membranes for 1 hour. Then, membranes were blocked 1 hour at room temperature with TBS (Tris buffered saline solution) and 5% BSA, and blotted overnight in TBS 5% BSA containing primary antibodies (1:500; anti-p53 [rabbit, Santa Cruz Biotechnology Inc., sc-6243], -MRE11 [rabbit, Novus Biologicals, NB100-142], -LSD1 [mouse, Santa Cruz Biotechnology Inc., sc-271720]). After extensive washes in TBS 0.1% Tween-20, membranes were incubated 1 hour at room temperature with HRP-conjugated secondary antibodies at 1:5000 before being revealed with Enhanced chemo-luminescence (ECL). Acquisition was performed with a Fusion FX7 imager (Vilber-Lourmat).
6
Quantitative Analysis of Protein-Protein Interactions
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The protein samples were electrophoresed in the 6%–15% gradient polyacrylamide gels and blotted onto the PVDF membrane (Amersham Hybond P 0.45 PVDF blotting membrane, Cytiva). The proteins of interest were probed using the primary antibodies, that is, mouse anti‐Myc tag (Cell Signaling Technology), rabbit anti‐DDDDK (FLAG) tag (Genetex), rabbit anti‐histone H3 (R&D), rabbit anti‐CRABP2 (Proteintech), and peroxidase‐conjugated anti‐rabbit or anti‐mouse secondary antibodies. The proteins were then detected based on chemiluminescence (ECL Prime Western Blotting System, Cytiva) using Fusion FX7 imager (Vilber Lourmat). Regarding the densitometric analysis, immunoblot band intensities were quantified using the ImageJ software (Schneider et al., 2012 (link)). For the analysis of NR2F1 interaction with dimeric partners (NR2F1, NR2F2, and RXR) by conventional IP and GCE‐enabled photocrosslinking IP, the band intensity of the co‐IP Myc‐tagged partner was divided by the intensity of each FLAG‐NR2F1 variant (WT or mutants) (R1 = Myc‐partner/FLAG‐NR2F1). A final normalized intensity value (Rfin) presented in the plot was obtained by normalizing R1 of from each NR2F1 variant (R1var) against R1 from wild‐type NR2F1 (R1wt) (Rfin = R1var/R1wt, Rfin = 1 for NR2F1 WT).
7
Protein Extraction and Immunoblotting
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Total proteins were extracted from cell pellets corresponding to 2 to 5 ODs, as previously described45 (link). Briefly, cell lysis was performed on ice using 0.3 M NaOH and 1% beta-mercaptoethanol prior to protein precipitation with trichloroacetic acid (TCA) (7% final). Following full speed centrifugation, pellets were resuspended in HU loading buffer and heat-denaturated at 70˚C. Soluble fractions were recovered, and samples were analyzed by standard immunoblotting procedures using 1:3000 peroxydase-conjugated antiperoxydase (PAP, to detect protein-A-tagged proteins) (Sigma, #P1291, RRID:AB_1079562), 1:3000 monoclonal anti-FLAG antibody (Sigma, #F3165, RRID:AB_259529), 1:3000 anti-CDC2 antibody (Abcam, #ab5467, RRID:AB_2074778), 1:1000 anti-GFP antibody (Roche, # 11814460001, RRID: AB_390913), 1:500 anti-CBP antibody (Millipore, #07-482, RRID:AB_310653), 1:5000 goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, #sc-2005, RRID:AB_631736) and 1:5000 goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, #sc-2004, RRID:AB_631746). Detection was done with SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific, #34080), ECL Select reagent (GE Healthcare, #RPN2235), and a Vilber Lourmat Fusion Fx7 imager.
8
Exosomal MMP and TIMP Quantification
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Exosomal MMP and TIMP levels were measured using the Human MMP Antibody Array (Abcam), targetting 10 proteins, according to the manufacturer’s instructions. Exosome samples, previously isolated from 1 mL of conditioned media, were first lysed with RIPA 1X (Abcam) and resuspended in a total of 1 mL of PBS. Briefly, membranes were first blocked using the given Blocking Buffer and then incubated with the resuspended samples overnight at 4 °C. The following day, membranes were washed and incubated with the biotin-conjugated antibodies for 2 h at room temperature and then incubated in HRP-Conjugated Streptavidin overnight at 4 °C. Chemiluminescence was imaged using Fusion Fx7 imager (Vilber Lourmat), protein dots were quantified using ImageJ spot recognition analysis.
9
Quantitative Analysis of EDI3 and HER2 Expression
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Total RNA was isolated using the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions, and quantified using NanoDrop 2000. Two μg of total RNA was transcribed into cDNA with the High-Capacity cDNA Reverse Transcription Kit (Qiagen), and qRT-PCR performed with QuantiFast SYBR Green (Qiagen) with QuantiTect primer assays (Supplementary Table S2 A) on the ABI 7500 Fast Real-Time PCR system (Applied Biosystems). The relative expression of each target gene was calculated using 2−ΔΔCT.
For protein expression analysis, cells were lysed in RIPA buffer containing phosphatase and protease inhibitors (Sigma Aldrich), and the total protein concentration was measured using the BCA Protein Assay Kit (Thermo Fisher). Equal amounts of protein were separated on SDS polyacrylamide gels, transferred onto PVDF membranes, and protein expression was detected using antibodies listed in Supplementary Table S2 B. Images were taken on a Fusion Fx7 imager (Vilber) and bands quantified using ImageJ (National Institute of Health). In addition, EDI3 and HER2 protein expression was analysed with immunocytochemistry, as described in Supplementary Methods.
EDI3 activity was measured employing a modified version of the Amplex® Red Phospholipase D Assay Kit (Thermo Fisher Scientific), as previously described [11 (link)].
For protein expression analysis, cells were lysed in RIPA buffer containing phosphatase and protease inhibitors (Sigma Aldrich), and the total protein concentration was measured using the BCA Protein Assay Kit (Thermo Fisher). Equal amounts of protein were separated on SDS polyacrylamide gels, transferred onto PVDF membranes, and protein expression was detected using antibodies listed in Supplementary Table S
EDI3 activity was measured employing a modified version of the Amplex® Red Phospholipase D Assay Kit (Thermo Fisher Scientific), as previously described [11 (link)].
10
Western Blot Analysis of Protein Targets
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Proteins were resolved by SDS-PAGE, and transferred to nitrocellulose membranes (GE Healthcare) for 1 h. Then, membranes were blocked 1 h at room temperature with TBS (Tris buffered saline solution) and 5% BSA, and blotted overnight in TBS 5% BSA containing primary antibodies Anti-Poly (ADP-Ribose) Polymer antibody (1:500, mouse, Abcam, ab14459), anti-GFP 1:500, (rabbit, Invitrogen, A-11122) anti-Flag (mouse, MilliporeSigma, F1804), anti-PARP1 (1:500, Cell Signaling, 9532), anti-NUPR1 (rabbit, homemade) or anti-β-actin (1:500, mouse, Sigma, A5316). After extensive washes in TBS 0.1% Tween-20, membranes were incubated 1 h at room temperature with HRP-conjugated secondary antibodies at 1:5000 before being revealed with Enhanced chemo-luminescence (ECL). Acquisition was performed with a Fusion FX7 imager (Vilber-Lourmat). Uncropped blots are available in the Supplementary Fig. 6 .
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