Ecl western blot detection reagent

Freshly isolated monocytes and MDM were lysed in radio immunoprecipitation assay buffer [150 mM NaCl, 50 mM Tris-Cl, pH 7.5, 1% Nonidet P-40, 0.5% sodium deoxicholate, 0.1% sodium dodecyl sulphate (SDS)] containing the complete protease inhibitor cocktail (Roche Molecular Biochemicals). Protein concentrations were determined by protein assay (Bio-Rad Laboratories). Cell lysates (10–20 μg per lane) were fractionated on 8-12% SDS-PAGE, electroblotted to nitrocellulose filters (Protran BA 85, Schleicher & Schuell), and probed with Abs anti-human A3A (rabbit polyclonal D23, Santa Cruz Biotechnology), anti-human A3G (goat polyclonal 109727, Abcam), anti-human SAMHD1 (rabbit polyclonal 366–380, Sigma-Aldrich), anti-human Mx2 (goat polyclonal N17, Santa Cruz Biotechnology) and anti-actin (mouse monoclonal Abs-5, BD Biosciences) as gel loading control. The anti p-SAMHD1 T592 Ab has been previously described [99 (link)]. SuperSignal West Femto Substrate (Pierce) and ECL Western blot detection Reagent (Amersham) were used according to the manufacturer’s instructions. Levels of A3A and Mx2 proteins were quantified using a GS-800 Calibrated Densitometer (Bio-Rad Laboratories).

Total proteins from cells and liver tissue were isolated using SDS lysis buffer (15 mM Tris/HCl, pH 6.8, 2.5% glycerol, 0.5% SDS, 1mM EDTA with protease complete cocktail). Equal amounts of protein (10-20 μg) were separated by 7.5 to 12.5 % SDS-PAGE, transferred onto nitrocellulose membranes, stained with appropriate primary (Supplementary Table 3) and secondary antibodies (Sigma, Deisenhofen, Germany) and visualized by ECL Western Blot Detection Reagent (Amersham Bioscience, Buckinghamshire, England).
Cell surface proteins were biotinylated using a biotinylation kit (Pierce, Rockford, IL, USA), precipitated using streptavidin coupled agarose beads and analyzed by Western blot.
For immunofluorescence analysis, cells were fixed with 100% ice-cold methanol and stained with appropriate primary (Supplementary Table 3) and secondary antibodies (Alexa Fluor™ 488 or 594 conjugate (Molecular Probes, Eugene, OR, USA)). Nuclei were stained with Diamino-2-phenylindol (DAPI). Fluorescence images were acquired using confocal microscope FluoView1000 (Olympus, Hamburg, Germany).

Proteins were separated by electrophoresis in 12% and 6% polyacrylamide gels according to molecular weight. Then, they were transferred to 0.2 μm nitrocellulose membranes (Bio-Rad) using transfer buffer (25 mM Tris, 190 mM glycine and 20% methanol). Membranes were blocked for 60 min at 37 °C with 3% bovine serum albumin (BSA) in TBST-1X (150 mM NaCl, 20 mM Tris, 0.1% Tween-20, at pH 7.5) and incubated overnight at 4 °C with mouse anti-tubulin (1:2,000 Cell Signaling), mouse anti-GOLPH3 (1:1,000 Santa Cruz), rabbit anti-H2A.X (1:500, Cell Signaling), mouse anti-TGN38 (1:500 Santa Cruz), rabbit anti-phospho AKT1 (1:1,000, Cell Signaling), and rabbit anti-phospho mTOR (1:1,000 Cell Signaling) antibodies. After washing, membranes were incubated with anti-rabbit or anti-mouse secondary antibodies (1:2,500) (Jackson ImmunoResearch). Chemiluminiscent detection of immunodetected bands was performed using the ECL Western blot detection reagent (Amersham).

NDM-1 expression strain E. coli BL21(DE3) (pET28a-NDM-1) and E. coli ZC-YN3, K.pneumoniae QD-KP2 were cultured in LB medium supplemented with isoliquiritin (0, 16 and 64 μg/mL) at 37 °C with shaking for 6 h. The cultures were centrifuged at 12,000 rpm for 5 min, collected culture supernatant and bacteria for preparing samples for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. Then, protein was transferred onto polyvinylidene fluoride (PVDF) membranes, blocked with 5% nonfat milk for 2 h at room temperature incubated with anti-NDM-1 mouse polyclonal antibody for 2 h, correspondingly, and then used HRP-conjugated goat anti-mouse antiserum for incubation for 1 h; then, the blots were tested with Amersham ECL Western Blot Detection Reagent.

To determine infection of primary cultures, cells were lysed in lysis buffer
(25 mM Tris pH 7.5, 1% TritonX-100 and 1% β-octyl
glucoside). 50μg of protein was treated with
3.75 μg/ml of proteinase K at 37°C for
30 minutes and methanol-precipitated prior to resuspension in
SDS-loading dye and running on a 12% Tris-Glycine gel. Western blots were
carried out with Sha31 antibody (SPIBio, mouse anti-PrP, 1:5000),
β3-tubulin (Sigma Aldrich, mouse 1:5000), a-tubulin (Sigma-aldrich,
mouse 1:10000), GFAP (Dako, rabbit 1:5000). Peroxidase-conjugated secondary
antibodies to mouse or rabbit were used (GE Healthcare) and blots were revealed
with ECL Western Blot detection reagent (Amersham). For assays on conditioned
medium (CM), cells were plated in T-25 flasks and grown to confluence. Medium
was removed, cells were washed twice with PBS and 3 ml of serum-free
media added. After 24 h conditioning, medium was removed and
pelleted at 2000 rpm to remove debris. Cells were lysed in
1 ml lysis buffer. 1.5 ml CM was used for detecting PrP
in the absence of PK treatment. The other 1.5 ml was precipitated in
3 vols of methanol overnight
(−20 °C), pelleted at 6000 rpm
for 15 minutes and the protein pellet resuspended in
50 μl of lysis buffer. Protein in lysates and
re-solubilised CM were quantified and PK resistance determined as described
above.

HRECs obtained from passage 6 were grown to 70–80% confluence and then starved for 12 h in 0.5% FBS/ECM. HRECs were pretreated with Mcc950 for 2 h before stimulation with high glucose. Then, HRECs and proliferative membrane samples from DR patients or donor eyes were lysed using a nuclear and cytoplasmic protein extraction kit (Beyotime, Haimen, China). Lysates were centrifuged at 15 000 × g for 10 min at 4 °C. Protein was quantified using Bradford’s reagent with bovine serum albumin as a standard. Proteins were separated using 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, IPVH00010, Bedford, MA, USA). Then, the membrane was blocked for 1 h at room temperature with 5% (v/v) nonfat dry milk. After three washes with PBST, the membrane was incubated in PBS at 4 °C (overnight) with anti-caspase 1 (1:1000, Cell Signaling Technology, Boston, MA, USA, No.2225), anti-NLRP3 (1:1000, Proteintech, Chicago, USA, No. 19771-1-AP), anti-IL-1β (1:1,000, Cell Signaling Technology, No. 12242). The membrane was again washed with PBST and incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibodies. Signals were developed using a standard ECL western blot detection reagent (Amersham Biosciences, Arlington Heights, IL, USA). Densitometric analysis was performed with ImageJ software.

Protein lysates were isolated from liver tissues and primary mouse hepatocytes using a radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Elpis-Biotech, Dajeon, Republic of Korea). Proteins from whole-cell extracts were separated using 10% SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA), as described previously^{23 (link),26 (link)}. The blotted membranes were probed with BTG2 (1:1000), YY1 (1:1000), PCK1 (1:1000), β-actin (1:3000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and/or G6PC (1:1000, Abcam, Cambridge, UK) antibodies. After incubation with the indicated antibodies, immunoreactive proteins were developed with an ECL Western Blot Detection Reagent (Amersham Biosciences).