Thioglycollate media (Remel, #R07178) was injected i.p. into male WT and Clec2d−/− mice for 3-5 days to elicit PECs. PECs were collected by lavage of the peritoneum with 8 ml/mouse RPMI-2%FBS, washed once with HCM, and then allowed to attach to tissue culture dishes for 30 min at 37°C. After washing off non-adherent cells, the dishes were incubated with 5 mM EDTA/PBS for 15 min to loosen the adherent macrophages. The cells were then pipetted off the dish with complete RPMI. The purity of macrophages (CD11b+, F4/80+) was greater than 95% (data not shown). The macrophages were added to 12-well plates at 2x106/well and incubated for 4h before stimulating them without or with 30 μg/ml purified histones or 40 μg/ml Zymosan for 30 min. The cells were then washed with PBS and lysed with 120 μl of RIPA buffer (Boston Bioproducts, #BP-115) containing protease inhibitors (Roche, #11697498001) and phosphatase inhibitors (Sigma, #P5726), followed by mixing with 4X LDS sample buffer (Invitrogen, #NP0008) with reducing agent (Invitrogen, #NP0009) and heating at 95°C for 10 min. Western blot was carried out to detect phospho-MAPKs (p38, ERK, JNK), phospho-Syk, and phospho-NFκB (p65), as well as total MAPKs and Syk.
Np0009
Manufactured by Thermo Fisher Scientific
Sourced in United States, Switzerland
The NP0009 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed to perform a core function within the laboratory setting. No further details about the intended use or specific capabilities of the NP0009 can be provided in an unbiased and factual manner.
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Lab products found in correlation
Np0007, by Thermo Fisher Scientific (15 mentions)
Anti flag m2 magnetic bead, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Supersignal west femto ecl substrate, by Thermo Fisher Scientific (3 mentions)
Ibright fl1500 imaging system, by Thermo Fisher Scientific (3 mentions)
Np0336box, by Thermo Fisher Scientific (3 mentions)
Las 4000, by Cytiva (2 mentions)
Np0002, by Thermo Fisher Scientific (2 mentions)
Np0321pk2, by Thermo Fisher Scientific (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Image studio software, by LI COR (2 mentions)
Alpha tubulin ab80779, by Abcam (2 mentions)
Psmc2 mss1 104, by Enzo Life Sciences (2 mentions)
Psmd1 c 7, by Santa Cruz Biotechnology (2 mentions)
Beta actin n 21, by Santa Cruz Biotechnology (2 mentions)
Erα f 10, by Santa Cruz Biotechnology (2 mentions)
Odyssey clx imaging machine, by LI COR (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
25 protocols using np0009
1
Macrophage Activation Signaling Assay
2
RhoA Activity Assay in Cultured Cells
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Fifteen Million cells were plated on regular plastic, collagen-coated or laminin-coated plates and incubated for 3 h at 37°. Cells were washed with PBS and lysed with the lysis buffer included in the RhoA activity assay kit (Cytoskeleton, BK036). Lysates were incubated with 30 µG RhoThekin beads for 1 h at 4° while rotating. Samples were washed once with wash buffer and 20 µL of 2× sample buffer (Invitrogen, NP0007 and NP0009) was added. Samples were incubated at 95° for 5 min, run on NuPAGE™ 4–12% Bis-Tris Protein Gels (Thermo Fisher, NP0321), and transferred overnight on 0.2 µM nitrocellulose membranes (Bio-rad, 1620112) in transfer buffer (25 mM Tris, 2 M glycine, 20% methanol in demineralized water). Membranes were blocked in 5% nonfat dry milk in TBS-T (pH 7.6, 20 mM Tris, 137 mM NaCl, 0.005% Tween-20 in demineralized water). Mouse anti RhoA antibody (1:500, included in kit) was incubated overnight in 0.5% nonfat dry milk in TBS-T. Membranes were washed three times in TBS-T and incubated with the secondary antibody rabbit anti-mouse HRP (1:2000; Dako, P0260) in 5% nonfat dry milk in TBS-T. Stained membranes were washed three times for 10–20 min in TBS-T and subsequently developed using ECL (Pierce, 32209). The signal intensities were measured by densitometry in ImageJ and normalized to actin loading controls.
3
Immunoblotting Analysis of Viral Proteins
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The virus-infected MDCK or VeroE6/TMPRSS2 cells in 6-well plates were washed with PBS and lysed in 500 μl of 1× TNT buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol). Lysates were centrifuged at 20,630 × g for 10 min at 4 °C. The supernatant was mixed with 4× lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, NP0007) and 10× sample reducing agent (Invitrogen, NP0009). Samples were boiled for 5 min and fractionated by NuPAGE 10% Bis-Tris Protein Gels (Invitrogen, NP0316BOX) and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, 170-4156). The membranes were incubated with mouse anti-influenza A virus NS1 (Santa Cruz, sc-130568; 1:1000), mouse anti-influenza A virus M2 (Santa Cruz, sc-32238; 1:1000), mouse anti-α tubulin (Santa Cruz, sc-32293; 1:2000), or rabbit anti-SARS-CoV-2 nucleocapsid (Cell Signaling, 33336; 1:1000) antibody, followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG (Jackson Immuno Research Laboratories, 115-035-003; 1:10,000) or anti-rabbit IgG (Invitrogen, G-21234; 1:10,000). The PVDF membranes were then treated with Chemi-Lumi One Super (Nacalai Tesque, 02230-30) to elicit chemiluminescent signals, which were detected and visualized using an LAS-4000 Mini apparatus (GE Healthcare).
4
Immunoprecipitation of RISC Complex Proteins
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Cells were lysed in SBB buffer (1% Triton X-100, 150mM NaCl, 50mM Tris-Cl at pH 7.5, 1mM EDTA) supplemented with protease inhibitors (Sigma, P8340) and phosphatase inhibitor (Sigma, P5726). Cell lysates were incubated at 4°C for 30 min and centrifuged at 12,000 g for 10 min at 4°C. Lysates were incubated with anti-Drosha, anti-Ago2, anti-Dgcr8, and anti-IgG (negative control) nutating overnight at 4°C followed by the addition of Dynabeads™ Protein A/G (Invitrogen, 10002D/10004D) and rocking for 4 hr at 4°C. The magnetic beads were precipitated and rinsed with SBB buffer for 5 min at 4°C for three times, followed by adding sample buffer (Invitrogen, NP0007) with reducing agent (Invitrogen, NP0009) and heated at 95°C for 3 min.
5
Overexpression and Immunoprecipitation of Drosha
6
Overexpression and Immunoprecipitation of Drosha
7
Drosha Overexpression and Protein Interaction
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293T cells were transfected with pcDNA4-hDrosha and pCI HA NEDD4 or pCI empty vector. 2μg/ml doxycycline was added into medium to induce Drosha overexpression. 48 hours after transfection, cells were lysed in SBB buffer (1% Triton X-100, 150mM NaCl, 50mM Tris-Cl at pH 7.5, 1mM EDTA) supplemented with protease inhibitors (cOmplete, Roche-11836170001) and PhosStop (Roche-04906845001). Lysates were incubated at 4˙C for 30 min, followed by centrifugation at 12,000 g for 10 min at 4˙C. 1/10 of the lysate was saved as input sample for immunoblot. The lysate was incubated with anti-Flag M2 Magnetic Beads (sigma, M8823) and rocked overnight at 4˙C. M2 beads were then washed in SBB buffer for 5 min at 4˙C for three times, and boiled in sample buffer (Invitrogen, NP0007) with reducing agent (Invitrogen, NP0009). The elute and input are subjected to immunoblot analysis.
8
In vitro Cleavage Assay for Neurodegenerative Proteins
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For in vitro cleavage assays, 1 µg of recombinant full-length human α-syn (Abcam #ab51189), TDP-43 (R&D #AP-190) or 4N2R tau (rPeptide #T-1001–1) was incubated with or without 1 µM of each protease. Proteases requiring pre-activation were performed, as mentioned in Table S2 . The following buffers were used as indicated: 100 mM sodium citrate pH 3.4, 50 mM sodium acetate pH 4.5 or 5.5 or 100 mM phosphate buffer saline (PBS) pH 7.4. Also, 1 mM EDTA and 2 mM DTT were used and each reaction was performed over 1 h at 37ºC. The assay was performed in a total volume of 19.5 µl. Protease activity was stopped by adding 7.5 µl of NuPAGE 4X LDS (Fisher Scientific #NP0007) and 3 µl of 10X reducing agent (i.e., 50 µM) (Fisher #NP0009). Samples were then immediately denatured for 10 min at 80ºC. All samples were run on precast NOVEX 4–12% Bis–Tris gels (Fisher #NP0321PK2) using MES buffer (Fisher #NP0002). The gel was then either fixed in 40% ethanol and 10% acetic acid for silver stain or transferred onto nitrocellulose membranes for Western blotting. Silver staining was performed according to the manufacturer’s instructions (Thermo Fisher #LC6070).
9
Progranulin Cleavage by Recombinant AEP
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400 ng of recombinant human progranulin (R&D #CF-2420) was incubated at 37 °C for 60 min with recombinant human AEP (R&D #2199-CY) at pH 4.5. The cleavage was stopped by adding 7.5 μl of NuPAGE 4X LDS (Fisher #NP0007), 3 μl of 10X reducing agent (Fisher #NP0009) and denatured for 10 min at 70 °C. The samples were run on precast NOVEX 4–12% Bis-Tris gels (Fisher #NP0321PK2) using MES buffer (Fisher #NP0002). The gel was fixed, and silver stained as described above. Protein bands were excised and digested in-gel with Endoproteinase Asp-N (Sigma #11054589001) as described previously [25 (link), 26 (link)]. The extracted digests were vacuum-evaporated, resuspended in 20 μl of 0.1% formic acid, and desalted using C18 ZipTips (Millipore #ZTC18M096).
10
Cdkl5 Protein Interactome Mapping
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