Cells were incubated for 48 h in the presence of RAM2061. PI (bortezomib or carfilzomib) was added either concurrently or sequentially (24 h after RAM2061). Cells were washed with PBS and incubated with calreticulin polyclonal antibody (1:200, Invitrogen PA3-900), followed by secondary anti-rabbit Alexafluor594. Ten-thousand cell events were recorded using a BD LSRII flow cytometer. FlowJo software was used for all data analysis.
Pa3 900
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PA3-900 is a laboratory instrument designed for conducting analytical procedures. It features a compact and durable construction to enable reliable performance in various laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Co ip assay kit, by Thermo Fisher Scientific (1 mentions)
Clathrin heavy chain, by Abcam (1 mentions)
Lamp1, by Cell Signaling Technology (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Caveolin 1, by Cell Signaling Technology (1 mentions)
A11035, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Ab2907, by Abcam (1 mentions)
Ab10287, by Abcam (1 mentions)
Sc 53711, by Santa Cruz Biotechnology (1 mentions)
Ff26a f9, by BioLegend (1 mentions)
Fv1200 laser scanning confocal microscope, by Olympus (1 mentions)
Ab179513, by Abcam (1 mentions)
Serca2, by Cell Signaling Technology (1 mentions)
Sc 6217, by Santa Cruz Biotechnology (1 mentions)
Calnexin, by Merck Group (1 mentions)
Calreticulin, by Thermo Fisher Scientific (1 mentions)
Phospho eif2α, by Cell Signaling Technology (1 mentions)
12 protocols using pa3 900
1
Evaluating Calreticulin Exposure in RAM2061-Treated Cells
2
Co-IP Assay for RCN2-CALR Interaction
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The Co-IP experiments were performed to determine the interaction between RCN2 and CALR using a Co-IP assay kit (#26,146; Thermo Fisher Scientific). CNE2 and 5-8 F cells were lysed in 500 µL solubilization Co-IP buffer. We use 10–50 µg of protein for positive control. Then, 30 µL of RCN2 (10193-2-AP; Proteintech, Illinois, USA) or 26 µL of CALR (PA3-900; Invitrogen) were added to the rest of the supernatant for batch binding. After the beads were collected and washed 5 times, we eluted the beads with 200 µL of spent regenerant.
3
Intracellular Trafficking of Nanoparticle-Mediated Gene Delivery
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Cells were grown to 50-70% confluency on coverslips in 24-well plates and subjected then to different experimental immunofluorescence analyses. To assess the intracellular uptake and trafficking of nanosystems, cells were treated with 0.44 mg mL -1 PSN-pDNA (TopFluor-labelled PSN/Cy5-labelled pDNA) for different time points. Specifically, to test the intracellular trafficking, cells were treated with PSN-pDNA for 4 h then medium was replaced, and cells were cultured on complete medium for 2 and 4 h. After, cells were fixed with 4% paraformaldehyde for 20 min at RT followed by 5 min permeabilization with 0.1% Triton™ X-100. Then, coverslips were incubated with corresponding primary antibodies, caveolin-1 (3267, Cell Signalling Technology, Massachusetts, USA), clathrin heavy chain (ab21679, Abcam, Cambridge, UK), EEA1
(3288, Cell Signaling Technology), LAMP1 (9091, Cell Signaling Technology), calreticulin (PA3-900, Invitrogen) or GM130 (610822, BD Biosciences). After, incubation with secondary antibody was carried out using Alexa Fluor 546-labelled (A-11035, Invitrogen) for 1 h at RT. Cell nuclei were stained with DAPI (D1306, Invitrogen). Images were captured on a LSM710 confocal microscope (ZEISS, Oberkochen, Germany). The analysis and quantification were done using ImageJ software.
(3288, Cell Signaling Technology), LAMP1 (9091, Cell Signaling Technology), calreticulin (PA3-900, Invitrogen) or GM130 (610822, BD Biosciences). After, incubation with secondary antibody was carried out using Alexa Fluor 546-labelled (A-11035, Invitrogen) for 1 h at RT. Cell nuclei were stained with DAPI (D1306, Invitrogen). Images were captured on a LSM710 confocal microscope (ZEISS, Oberkochen, Germany). The analysis and quantification were done using ImageJ software.
4
Immunoprecipitation and RNA Extraction
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Cells were lysed by ice-cold RNA-IP lysis buffer (150 mM KCl, pH 7.4 25 mM Tris, 5 mM EDTA and 0.35% Triton X-100) containing 1× protease inhibitor cocktail. Protein concentrations were determined using Bradford assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Lysates containing 400 µg total protein were pre-cleaned with PureProteome™ Protein A/G Magnetic Beads (Invitrogen, Waltham, MA, USA) at 4 °C for 1 h. The pre-cleaned lysates were incubated with rabbit anti-CRT antibody (PA3-900, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C overnight. The protein–antibody complex was pulled down by magnetic beads at 4 °C for 4 h. Then, all the beads were collected and washed with ice-cold RNA-IP lysis buffer for three times, followed by three washes with NT2 buffer (50 mM pH 7.4 Tris, 150 mM NaCl and 0.05% NP-40). For Western blot, the beads were mixed with sample buffer and then boiled at 100 °C for 15 min for protein elution. For real-time PCR, beads were subjected to RNA extraction by TRIzol reagent.
5
Comprehensive Antibody Panel for Cell Surface and Intracellular Antigens
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Antibodies for flow cytometric detection of surface antigens: α2-integrin (P1E6-C5), α3-integrin (ASC-1), α4-integrin (9F10), α5-integrin (NKI-SAM-1), α6-integrin (GoH3), F4/80 (BM8, BioLegend); β1-integrin (sc-53711, Santa Cruz); CRT (ab2907), ERp57 (ab10287, Abcam). Antibodies for flow cytometric detection of intracellular antigens: CRT (ab2907), PDI (ab2792) and cytochrome C (6H2.B4, Biolegend). Antibodies for immunoprecipitation and immunoblotting: α4-integrin (HP2/1), CRT (PA3-900, ThermoFisher), ERp57 (ab10287, Abcam), and GAPDH (FF26A/F9, BioLegend). Antibodies for integrin activation and phagocytosis assays: β1-integrin (9EG7) and CD47 (B6H12, BD Biosciences).
6
Evaluating Transcription Factor-ER Colocalization
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MDA-MB231 cells were co-transfected with pEGFP-Fra-1 or pEGFP-c-Fos and both NA deletion mutants fused to the HA tag or the control with the empty vector. Immunofluorescence was performed using anti-HA (mouse Sigma #H9658 1:100) and anti-calreticulin (rabbit Thermo Scientific #PA3-900 1:800), secondary antibodies: anti-mouse 546 and anti-rabbit 643 (1:600). A spectral Olympus (Center Valley, PA) FV 1200 confocal laser scanning microscope with a plan apochromatic 60x (SC), numerical aperture 1.4, oil immersion objective lens zoomed digitally 5x was used. Mander's colocalization coefficient M1 (proportion of Fra-1 or c-Fos present in the ER) with threshold was used to quantify colocalization using the Fiji software and the Jacop plugin.
7
Protein Expression Detection Methods
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Expression of proteins were detected using antibodies against mouse lamin A/C (SAB4200236, Sigma‐Aldrich, St. Louis, MO, USA), human lamin A (MAB3540, Millipore, Burlington, MA, USA), Sarcolipin (ABT13, Millipore), β‐tubulin (ab179513, Abcam, Cambridge, UK), FLAG (F7425, Sigma‐Aldrich), HA (H6908, Sigma‐Aldrich), lamin B1 (ab133741, Abcam), lamin B (sc‐6217, Santa Cruz, Dallas, TX, USA), Calnexin (MAB3126, Millipore), Calreticulin (PA3‐900, Thermo Fisher Scientific), SERCA2 (4388, Cell Signaling, Danvers, MA, USA), β‐ACTIN (A5441, Sigma‐Aldrich), Grp78 (ab108613, Abcam), Chop (2891, Cell Signaling), eIF2α (2103, Cell Signaling), phospho‐eIF2α (3398, Cell Signaling), Atf4 (SC‐200, Santa Cruz), IRE1α (3294, Cell Signaling), phospho‐IRE1α (PA1‐16927, Thermo Fisher Scientific), and Gapdh (GTX100118, GeneTex, Hsinchu, Taiwan).
8
Quantification of CRT Levels in Cell Supernatants
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CRT released in to 1 ml of culture media from ~ 1 × 105 cells treated with DX and/or TG with or without TUDCA was measured by ELISA [30 (link)]. Briefly, 153 μl of cell-free supernatant was placed into wells of a 96-well ELISA plate with 17 μl of 10 × carbonate buffer, pH 9.6. The protein supernatants were left to bind at 4 °C overnight. The plates were then washed four times with phosphate-buffered saline (PBS) containing 0.1% v/v Tween 20 (PBST). Remaining binding sites were blocked with 5% v/v BSA in PBST at 37 °C for 30 min. Wells were washed a further four times with PBST. Next 100 μl of 1:2000 dilution of rabbit anti-human CRT antibody (Thermofisher PA3-900) diluted in PBST was added to each well for 2 h in 37 °C. The wells were washed again as described above, and a 100 μl of 1:2000 dilution of secondary anti-rabbit HRP-conjugated antibody was added to the wells and the plate incubated 1 h in 37 °C. The reaction was developed for 15 min at RT in the dark and was terminated by adding 50 μl 2N H2SO4 to each well. The optical density at 450 nm (OD45nm) of each sample was measured on a BMG Labtech FLUROstar™ plate reader. A CRT standard curve was generated from known concentrations of CRT (Fig. S3).
9
Multicolor Flow Cytometry Antibody Panel
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Rabbit polyclonal antibody against calreticulin (PA3-900) was purchased from ThermoFisher (Rockford, IL, USA). Alexa Fluor 488 goat anti-rabbit IgG (A11008) was purchased from Life Technology (Eugene, OR, USA). PE-conjugated anti-mouse CD4 (clone RM4-5),PE/Cy7-conjugated anti-mouse CD8 (clone 53-6.7), FITC-conjugated anti-mouse CD25 (clone 3C7), Alexa Fluor 488-conjugated anti-mouseFoxP3 (clone 150D), APC-conjugated anti-mouse Gr-1 (clone RB6-8C5) and FITC-conjugated anti-mouse CD11b (clone M1/70) antibodies were purchased from BioLegend (San Diego, CA). PerCP-eFluor 710-conjugated anti-mouse CD3 (clone 17-A2)and APC eFluor 780-conjugatedanti-mouse CD127 (clone A7R34) antibodies were purchased from eBioscience (San Diego, CA).
10
Immunoblotting for HA-tagged and ER Proteins
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Unless otherwise stated, the cells were lysed in Triton X-100 lysis buffer (1% [vol/vol] Triton X-100, 25 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM EGTA containing complete EDTA-free protease inhibitor). Protein concentrations were determined using the Pierce BCA assay. Proteins were separated by 15% SDS–PAGE and transferred to PVDF membranes. Membranes were blocked in 5% milk in PBST (PBS with 0.1% [vol/vol] Tween 20) and incubated with antibodies diluted in PBST, either for 1 h at RT or overnight at 4°C. The following primary antibodies were used: rat anti-HA (clone 3F10, 1:1,000; Roche), mouse anti-HA (ab18181, 1:1,000; Abcam), rabbit anti-calreticulin (PA3-900, 1:5,000; Thermo Fisher Scientific), and anti-calnexin (CANX) (ab22595, 1:1,000; Abcam). For blots imaged using an Odyssey system (Li-COR), goat antirat IRDye 800 secondary (1:10,000) or goat antirabbit IRDye 680 (1:10,000) was used. For ECL detection, HRP-conjugated secondary antibodies and Luminata HRP substrate (WBLUR0100; Millipore) were used.
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