Cells were cultured in 6-well plates and treated with drugs for 48 hours. The cells were subsequently harvested and stained with antibodies to detect intranuclear/intracellular protein levels. The antibodies were purchased from Abcam (UK) unless otherwise indicated: rabbit anti-CBL and rabbit IgG isotype control (Cell Signaling Technologies) detected with anti-rabbit IgG-PE (Cell Signaling Technologies); mouse anti-p21 and mouse IgG2b isotype control detected with anti-mouse IgG-Alexa Fluor 488; rabbit anti-TNFAIP3-Alexa Fluor 488 and rabbit IgG isotype control-Alexa Fluor 488; rabbit anti-HO-1-Alexa Fluor 568 and rabbit IgG isotype control-Alexa Fluor 568. Data was acquired with a BD Accuri (BD Biosciences) and analysed with FlowJo software.
Rabbit igg isotype control
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Rabbit IgG isotype control is a laboratory reagent used as a control in immunological experiments. It is composed of immunoglobulin G (IgG) antibodies derived from rabbit serum. This reagent serves as a non-specific control to establish baseline signals and account for non-specific binding in assays that utilize rabbit-derived primary antibodies.
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Lab products found in correlation
Mouse igg isotype control, by Cell Signaling Technology (4 mentions)
Anti rabbit igg pe, by Cell Signaling Technology (2 mentions)
Leupeptin, by Merck Group (2 mentions)
Aprotinin, by Merck Group (2 mentions)
Igepal ca 630, by Merck Group (2 mentions)
Accuri, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Anti α6 integrin pe goh3, by BD (1 mentions)
Alexa fluor 647 mouse anti sox2, by BD (1 mentions)
Rabbit anti c myc, by Cell Signaling Technology (1 mentions)
Rabbit anti lin28, by Cell Signaling Technology (1 mentions)
Apc anti plzf antibody, by R&D Systems (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
Mg132, by Merck Group (1 mentions)
Anti myc, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti usp21, by Thermo Fisher Scientific (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
20 protocols using rabbit igg isotype control
1
Flow Cytometric Analysis of Intracellular Proteins
2
Isolation and Characterization of Testicular Stem Cells
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Testicular single-cell suspensions were prepared from 2–3-month-old Col1a1-4F2A-OSKM mice as described previously [34 ]. The immunomagnetic selection of α6+ cells was performed using anti-α6 integrin-PE (GoH3) (BD Pharmingen) and anti-PE microbeads (Miltenyi Biotec) according to the manufacturer's protocol. Hoechst staining (5 μg/ml) of the cell suspensions was performed as described previously [34 ]. Cells were then labelled with β2m-FITC (Santa Cruz) and anti-CD117-APC (2B8) antibodies (BD Pharmingen). Propidium iodide (Sigma) was added before cell sorting to exclude dead cells. To analyse the transcription factors, cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Pharmingen) and stained with the following antibodies: Alexa Fluor 488 mouse anti-Nanog (BD Pharmingen 560261 clone M55-312), Alexa Fluor 647 mouse anti-Sox2 (BD Pharmingen 560294 Clone 245610), rabbit anti-c-Myc (Cell Signaling), rabbit anti-Kfl4 (Abcam), rabbit anti-Lin28 (Cell Signaling), rabbit IgG isotype control (Cell Signaling) and secondary anti-rabbit Alexa Fluor 488 antibody along with APC-anti-PLZF antibody (R&D). Analyses and cell sorting were performed on ARIA, LSR II and FACSCalibur flow cytometers (Becton Dickinson).
3
Comprehensive Immunoblotting Protocol
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The following reagents and antibodies were used in this study:
MG132 (Sigma-Aldrich, Cat#M7449); anti-Flag (Sigma-Aldrich, Cat#F7425); anti-MYC (Sigma-Aldrich, Cat#M4439); anti-STAT3 (Cell Signaling Technology, Cat#9319S); anti-phospho-STAT3 (Cell Signaling Technology, Cat#9145S).
Mouse IgG Isotype control (Cell Signaling Technology, Cat#53484).
Rabbit IgG Isotype control (Cell Signaling Technology, Cat#2729); anti-Usp21 (Invitrogen, Cat#PA5-110556); anti-beta actin (2D4H5) (Proteintech, Cat#66 009-1-Ig).
MG132 (Sigma-Aldrich, Cat#M7449); anti-Flag (Sigma-Aldrich, Cat#F7425); anti-MYC (Sigma-Aldrich, Cat#M4439); anti-STAT3 (Cell Signaling Technology, Cat#9319S); anti-phospho-STAT3 (Cell Signaling Technology, Cat#9145S).
Mouse IgG Isotype control (Cell Signaling Technology, Cat#53484).
Rabbit IgG Isotype control (Cell Signaling Technology, Cat#2729); anti-Usp21 (Invitrogen, Cat#PA5-110556); anti-beta actin (2D4H5) (Proteintech, Cat#66 009-1-Ig).
4
Immunostaining of Anti-CD3–stimulated CD8+ T Cells
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Anti-CD3–stimulated CD8+ T cells were stained for confocal microscopy. In brief, cells were fixed in 2% paraformaldehyde on superfrost plus slides. Cells were washed 3 times in PBS-Triton X-100 0.1%, and then blocked with PBS containing BSA and glycin for 30 min before incubation with primary antibodies: anti-VEGFR2 (55B11; Cell Signaling Technology) or anti-CD8 (clone 53–6.7; eBioscience), or isotype control antibodies (rabbit IgG isotype control (Cell Signaling Technology) and rat IgG2a isotype control (eBioscience). Cells were then washed with PBS Triton 0.1% and incubated with secondary antibodies: goat anti–rabbit Alexa Fluor 647 and goat anti–rat Alexa Fluor 488 (Life Technologies). After washing, cells were incubated with DAPI and mounted over Fluoromount G (Interchim). Confocal microscopy was performed using the SP8 Leica microscope using LAS AF software (Leica) and ImageJ software.
5
Flow Cytometric Analysis of ULBP1 and NKG2D Interactions
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Intracellular and cell surface ULBP1s were detected by a flow cytometer. Briefly, after digitonin‐permeabilization of cells, intracellular ULBP1 was immunostained by using anti‐ULBP1 rabbit polyclonal antibody (GTX123021; GeneTex) and PE‐conjugated goat anti‐rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) as primary and secondary antibody, respectively. Cells without digitonin‐permeabilization were also subjected to the immunostaining of cell surface ULBP1. Rabbit IgG isotype control (Cell Signaling Technology) was used as a negative control. The mean fluorescence intensity and the standard deviation were calculated from four independent experiments.
The binding of NKG2D to target cells was also detected by a flow cytometer. Briefly, target cells were subjected to the treatment of recombinant human NKG2D/Fc (R&D Systems) or human IgG1/Fc chimera protein (R&D Systems), and then were immunostained by FITC‐conjugated goat anti‐human IgG, Fc fragment specific (Jackson ImmunoResearch Laboratories). The mean fluorescence intensity and the standard deviation were calculated from three independent experiments.
The binding of NKG2D to target cells was also detected by a flow cytometer. Briefly, target cells were subjected to the treatment of recombinant human NKG2D/Fc (R&D Systems) or human IgG1/Fc chimera protein (R&D Systems), and then were immunostained by FITC‐conjugated goat anti‐human IgG, Fc fragment specific (Jackson ImmunoResearch Laboratories). The mean fluorescence intensity and the standard deviation were calculated from three independent experiments.
6
Antibody Characterization for Chromatin Immunoprecipitation
7
Quantifying NFAT Transcription Factors
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Serial sections were selected at 80–100 μm intervals and 6–12 sections were stained for each sample. Tissue was treated with EDTA antigen retrieval solution pH 8.0 at 70 °C overnight and incubated with primary antibodies: mouse anti-human NFATc1 (clone 7A6, Santa Cruz), rabbit anti-human/mouse NFATc2 (clone D43B1, Cell Signaling Technology), mouse IgG1κ isotype control (Cell Signaling Technology), or Rabbit IgG isotype control (Cell Signaling Technology) at 4 °C overnight, followed by secondary antibodies according to a Polink-2 plus®Polymer HRP Detection System (ZSGB-Bio, Beijing, China). All sections were counterstained with hematoxylin.
For evaluation and semi-quantification of immunohistochemistry results, NFATc1 positive and total counterstained cells were counted, respectively, for each region of interest (ROI). Three ROIs were randomly selected for each section. The scoring system is based on the average percentage of NFATc1 positive in total cells [24 (link)]: 0, no positive cells; 1, less than 10% positive cells; 2, more than 10% but less than 50% positive cells; 3, more than 50% positive cells. All samples were scored by two independent researchers.
For evaluation and semi-quantification of immunohistochemistry results, NFATc1 positive and total counterstained cells were counted, respectively, for each region of interest (ROI). Three ROIs were randomly selected for each section. The scoring system is based on the average percentage of NFATc1 positive in total cells [24 (link)]: 0, no positive cells; 1, less than 10% positive cells; 2, more than 10% but less than 50% positive cells; 3, more than 50% positive cells. All samples were scored by two independent researchers.
8
Immunoprecipitation and Immunoblotting Protocol
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LNCaP cells were lysed with IGEPAL CA-630 buffer (50 mM Tris-HCl, pH 7.4, (Sigma, T5030), 1% IGEPAL CA-630 (Sigma, I8896), 10 mM EDTA, 150 mM NaCl, 50 mM NaF, 1 μM leupeptin (Sigma, L5793), and 0.1 μM aprotinin (Sigma, SRE0050)). Primary antibody was covalently immobilized on protein A/G agarose using the Pierce Crosslink Immunoprecipitation Kit according to the manufacturer’s instructions (Thermo Scientific, 26147). Samples were incubated with immobilized antibody beads for at least 2 h at 4 °C. Cell lysates were also subjected to immunoprecipitation with either mouse IgG isotype control (Cell Signaling Technology, 5415) or rabbit IgG isotype control (Cell Signaling Technology, 3900), depending on the immunoglobulin type of primary antibody. After immunoprecipitation, the samples were washed with TBS five times. They were then eluted with glycine-HCl (0.1 M, pH 3.5) and the immunoprecipitates were subjected to immunoblotting using specific primary antibodies.
9
Immunoprecipitation and Immunoblotting of Cardiomyocytes
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NRCMs or heart tissues were lysed with IGEPAL CA-630 buffer (50 mM Tris-HCl, pH 7.4, [Sigma-Aldrich, T5030], 1% IGEPAL CA-630 [Sigma-Aldrich, I8896], 10 mM EDTA, 150 mM NaCl, 50 mM NaF, 1 μM leupeptin [Sigma-Aldrich, L5793], 0.1 μM aprotinin [Sigma-Aldrich, SRE0050]). Samples were incubated for 2 h with primary antibody after being precleared with 30 μL PureProteome™ Protein A/G Mix Magnetic Beads (Merck Millipore, LSKMAGAG10) at 4 °C. Cell lysates were also subjected to immunoprecipitation with either mouse IgG1 isotype control (Cell Signaling Technology, 5415) or rabbit IgG isotype control (Cell Signaling Technology, 3900) according to the immunoglobulin type of primary antibody. After immunoprecipitation, the samples were washed five times with TBS. They were then eluted with glycine-HCl (0.1 M, pH 3.5) and the immunoprecipitates were subjected to immunoblotting using specific primary antibodies.
10
LARP1 RNA Immunoprecipitation and qPCR Analysis
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Cells were collected by trypsinization and re-suspended in RIP lysis buffer: 20 mM HEPES pH 7.4, 150 mM KCl, 5 mM MgCl2, 0.5% NP40, 400U/ml RNase inhibitor (Promega), 1 mM DTT, 400 μM VRC (NEB), protein (Roche) and phosphatase inhibitor (Calbiochem). Lysates were stored at −80°C overnight. RNA was immunoprecipitated with rabbit anti-LARP1 polyclonal antibody (SDIX-Novus Biologicals) or rabbit IgG isotype control (Cell Signalling Technology) following the method described by Keene et al. (26 (link)). RNA was extracted with Trizol (Life Technologies) and purified with the RNA clean-up and concentration micro kit (Norgen Biotek). To generate cDNA, immunoprecipitated RNA was reverse transcribed using the SensiScript® RT Kit (Qiagen) following the manufacturer's instructions. RT-qPCR was performed as described earlier. The fold enrichment for each target was measured by comparing the Ct values of LARP1-immunoprecipitated fraction to the IgG isotype fraction and normalized using the ΔCt formula.
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