Mice lungs were collected in DMEM and washed to remove any blood. The tissues were then dissociated using the Miltenyi Lung dissociation kit, following manufacturer’s instructions with slight modifications47 (link),48 (link). Briefly, after coarse digestion of lungs, 0.1 mg/mL soybean trypsin inhibitor (Sigma) 60 U/mL DNAse I and 1 mg/mL type XI collagenase was added and tissues were digested at 37 °C for 30 min. Then, lungs were finely homogenized in Miltenyi Gentle MACs, followed by addition of 0.1 M EDTA in FBS to inactivate the enzymes. Cell suspension was then passed through 70 μm filter, washed with PBS by centrifugation at 800×g for 10 min. RBCs were lysed using ACK lysis buffer (Alfa Aesar) and washed with DPBS. Cells were then counted and 1–2 million cells were stained with live/dead marker (Life Technologies) before being incubated with rabbit anti-V5 (1:250, Abcam ab9116) antibody followed by AlexaFluor 488 conjugated donkey anti-rabbit secondary antibody (2 μg/ml, Invitrogen A-21206). Cells were then fixed with BD Cytofix/Cytoperm (BD Biosciences) and acquired on a BD LSRFortessa flow cytometer. Compensation was performed using beads (BD Biosciences) labeled with fluorophore conjugated antibodies. Live cells were gated on total cells and analyzed for V5 positive cells. Data was analyzed using Flow Jo software (Treestar, Inc.).
Ab9116
Manufactured by Abcam
Sourced in United Kingdom
Ab9116 is a laboratory equipment product offered by Abcam. It serves as a core functional component, but a detailed unbiased description is not available.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Ab1791, by Abcam (2 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Soybean trypsin inhibitor, by Merck Group (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lung dissociation kit, by Miltenyi Biotec (1 mentions)
Live dead marker, by Thermo Fisher Scientific (1 mentions)
Ab37977, by Abcam (1 mentions)
Calnexin, by Santa Cruz Biotechnology (1 mentions)
Ubiquitin, by Cell Signaling Technology (1 mentions)
Lamin b, by Santa Cruz Biotechnology (1 mentions)
V9131, by Merck Group (1 mentions)
Mab1510, by Merck Group (1 mentions)
Ubi 1, by Merck Group (1 mentions)
Ab9354, by Merck Group (1 mentions)
Ab56889, by Abcam (1 mentions)
Sc 17764, by Santa Cruz Biotechnology (1 mentions)
F1804, by Merck Group (1 mentions)
Ab18184, by Abcam (1 mentions)
24 protocols using ab9116
1
Isolation and Flow Cytometric Analysis of Mouse Lung Cells
2
Western Blot Antibody Protocol
3
Western Blot and Immunofluorescence Antibodies
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The following primary antibodies were used for western blot (WB), immunofluorescence (IF) or meso scale discovery (MSD) assay: rabbit anti-PINK1 (WB, 1:2500; IF, 1:1000, #6946, D8G3), mouse anti-parkin (WB, 1:1000, #4211, Prk8, Cell Signaling Technology), mouse anti-Ub (WB, 1:2000, MSD, 1:500, MAB1510, ubi-1, Millipore), mouse anti-Mitofusin 2 (WB, 1:5000, ab56889, Abcam), mouse anti-TOM20 (IF, 1:100, sc-17764, Santa Cruz Biotechnology), rabbit anti-V5 (WB, 1:5000, ab9116, Abcam), rabbit anti-mCherry (WB, 1:2000, 5993-100, BioVision), rabbit anti-TUJ1 (WB, 1:1000, b3-tubulin, D71G9, Cell Signaling Technology), chicken anti-TUJ1 (IF, 1:500, β3-tubulin, AB9354, Millipore), mouse anti-GAPDH (WB, 1:150 000, H86504M, Meridian Life science), mouse anti-vinculin (WB, 1:250 000, V9131, Sigma-Aldrich). Rabbit anti-p-Ser65-Ub (1:5000 for WB, 1:250–500 for IF, and 1:250 for MSD) has been described recently (Fiesel et al., 2015a (link)).
4
Amyloid Precursor Protein and Presenilins Profiling
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The following primary antibodies were used: anti-APP CT (A8717, Sigma-Aldrich, St. Louis, MO); anti-β-amyloid, 17–24 (clone 4G8) (800701, BioLegend, San Diego, CA); anti-PS1 NT raised against the N terminus of PS1 (APS11) (ab15456, Abcam, Cambridge, MA); anti-PS1 CT raised against the C-terminus of PS1 (mAb5643, Cell Signaling Technology, Danvers, MA); anti-PS1 loop raised against the loop domain between transmembrane domains 6 and 7 of PS1 (E2000Y) (ab76083, Abcam, Cambridge, MA); anti-Syt1 (AB5600, Millipore, Temecula, CA); anti-Gapdh (mAb2118, Cell Signaling Technology, Danvers, MA); anti-MAP2 (ab5392, Abcam, Cambridge, MA); anti-His (ab18184, Abcam, Cambridge, MA); anti-V5 (ab9116, Abcam, Cambridge) and anti-FLAG M2 (F1804, Sigma-Aldrich, St. Louis, MO). Alexa Fluor 488 (ThermoScientific, Waltham, MA) and Cy3-labeled corresponding secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used for confocal microscopy imaging, and IRDye680/800- (Li-COR, Lincoln, NE) or HRP- (Jackson ImmunoResearch, West Grove, PA) conjugated ones were used for western blotting.
5
Immunohistochemistry of Synuclein and Neurotransmitters
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The following primary antibodies were used: mouse anti-α-Syn (BD610787, BD Biosciences, Franklin Lakes, NJ), rabbit anti-V5 (ab9116, Abcam, Cambridge, UK), mouse anti-α-Syn (LB509, Covance, Munich, Germany), rabbit anti-TH (Zytomed, Berlin, Germany), rabbit anti-TPH2 (ABN60, Millipore, Darmstadt, Germany), mouse anti-NeuN (MAB377, Millipore) and mouse anti-β-tubulin (Sigma, St- Louis, MO).
The following secondary antibodies were used: Cy2-conjugated goat anti-mouse, Cy3-conjugated goat anti-rabbit, Cy3-conjugated goat anti-mouse, Cy5-conjugated goat anti-rabbit (all from Jackson Immuno Research, West Grove, PA); horseradish peroxidase(HRP)-coupled goat anti-mouse and HRP-coupled goat anti-rabbit (both from Dianova, Hamburg, Germany).
The following secondary antibodies were used: Cy2-conjugated goat anti-mouse, Cy3-conjugated goat anti-rabbit, Cy3-conjugated goat anti-mouse, Cy5-conjugated goat anti-rabbit (all from Jackson Immuno Research, West Grove, PA); horseradish peroxidase(HRP)-coupled goat anti-mouse and HRP-coupled goat anti-rabbit (both from Dianova, Hamburg, Germany).
6
Co-immunoprecipitation of Bcl-6 Protein
7
Western Blot Analysis of Protein Expression
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Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Equal amount of protein lysate was resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Millipore) using fast Turbo Transfer (Bio-rad). After blocking in 5% skimmed milk for 1 h, the appropriate primary antibody was added: anti-ADAR2 (RED1) (1:1000, ab64830, Abcam), anti-SRp30c(SRSF9) (1:1000, sc-134036, Santa Cruz), anti-FLAG (1:2000, Clone M2, Sigma), anti-cMyc (1:1000, sc-789, Santa Cruz), anti-V5 (1:2000, ab9116, Abcam) or anti-β-actin (1:1000, clone C4, sc-47778, Santa Cruz). Primary antibodies were incubated overnight in the cold room. After washing with phosphate-buffered saline (PBS)/0.2% Tween-20 (PBST), a secondary antibody, namely horse-radish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5000, NA934V, Amersham) or HRP-conjugated anti-mouse IgG (1:5000, NA931V, Amersham), was added for 1 h. After washing with PBST, signals were detected using the WesternBright Sirius Chemiluminescent HRP Substrate (Advansta).
8
Western Blotting Antibody Sources
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The anti-BRM antibody used for Western blotting was raised in rabbit and has been characterized in a previous study [13 (link)]. An antibody raised against the rat BRG1 protein by Östlund-Farrants et al. [35 (link)] was used to detect BRM in S2 cells. The specificity of this antibody in D. melanogaster has been previously documented [13 (link)]. The antibodies against MOR and SNR1 were kindly provided by C. P. Verrijzer (see reference [36 (link)] and references therein). The monoclonal anti-alpha-tubulin antibody (clone B-5-1-2) was from Sigma-Aldrich and the anti-V5 antibodies were from Invitrogen (R96025) or Abcam (ab9116).
9
Western Blot Analysis of Protein Targets
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Cells were pelleted and lysed and equal protein amounts were subjected to SDS-PAGE and then transferred onto a nitrocellulose membrane. The following primary antibodies were used: anti-LPXN (ab181621, Abcam; LS‑C313296, LSBio), anti-ß-actin (A5441, Sigma Aldrich), anti-PYK2 (ab81266, Abcam), anti-GAPDH (2118S, CST), anti-phosphotyrosine (ab10321, Abcam), anti-V5 tag (ab9116, Abcam), anti-FAK antibody (3285, CST).
10
Antibody-based Characterization of DMXL2 and Notch
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We used two antibodies against DMXL2 (24415-1-AP from Proteintech) for western blot and immunofluorescence and ab122552 (abCam) for IHC and immunofluorescence. Antibodies against Notch1 and Notch3 were purchased from Cell Signalling (3608, and 2889) and Notch4 was purchased from Santa Cruz (Sc-5594 H-225). Actin and V5 antibodies were purchased from abCam (ab6276 and ab9116). ZEB1 (3396), ZO1 (8193), E-Cadherin (3195), and IQGAP1 (2293) antibodies were purchased from Cell Signalling. Bafilomycin A1 was purchased from Sigma (B1793). TBP was purchased from Santa Cruz. LysoTracker (LysoTracker® Red DND-99 Life Technologies) was used according to the manufacturer protocol for labelling acidic organelles in live cells following Bafilomycin A1 and DMXL2 siRNA treatments. In knock down experiments, the LysoTracker was added after 72 hours. Estradiol for culturing MCF7 was also purchased from Sigma and diluted in 100% Ethanol in a final concentration of 10−7M. Biotinylation assay was performed according to the manufacturer protocol (21435 Thermo Scientific). GSI was purchased from Pfizer, Inc (New York, NY, USA) (PF03084014). Dll4 ligand was purchased from abCAm (108557).
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