Nitrocellulose membrane

Protein extraction was performed using ice-cold RIPA buffer. Bradford assay (Bio-Rad) was used to assess protein concentration in the extracts. Proteins were resolved by electrophoresis in SDS-polyacrylamide gels (10%). Then, they were transferred to a nitrocellulose membrane (Macherey-Nagel, Düren Germany). The membrane was blocked for 1 h, at room temperature, in PBST with 5% nonfat milk and then with 5% bovine serum albumin incubated with primary antibodies overnight at 4 °C. Anti-PXR (1:500, sc-48403, Biotechnology Inc., Santa Cruz, CA, USA) and anti-b-Actin (1:1000 sc-8035; Biotechnology Inc., Santa Cruz, CA, USA) were used as primary antibodies. Horseradish peroxidase-conjugated secondary antibodies (Dako, CA, USA) were used at 1:5000 dilution. The detection of the immunoreactive bands was performed with the LumiSensor Chemiluminescent HRP Substrate kit (GenScript, NJ, USA). Relative protein amounts were evaluated by a densitometry analysis using ImageJ software. We used three independent samples for each group and β-actin for the normalization [40 (link)].

MSCs (5x10⁵ cells) were lysed in 200ul 1% SDS PBS. Cell extracts and supernatants were analyzed in SDS-PAGE electrophoresis followed by electrotransfer onto nitrocellulose membrane (Macherey-Nagel, Germany) using a semidry apparatus. Membranes were blocked with 5% w/v skim milk powder (Sigma Aldrich, USA) in PBST for 1h and incubated with mouse anti-GFP mAb (Millipore, MAB2510, 1:2000 dilution) or rabbit anti-TERT (Novus Biologicals, USA, NB120-32020, 1:2000 dilution) antibodies overnight. For loading controls, membranes were blotted with rabbit anti-GAPDH (Santa Cruz, USA, sc-25778, 1:1000 dilution) antibody. After incubation with the appropriate secondary alkaline phosphatase-conjugated antibody (Merck, Germany), membranes were developed with AP substrate NBT/BCIP (Applichem, Germany).

Total protein extracts of rosette leaves were prepared using the peqGOLD TriFast reagent (Peqlab). The protein concentration of the extracts was determined using the BCA protein assay kit (Pierce). Proteins were separated on 12–15% SDS-polyacrylamide gels and subsequently coomassie stained or blotted onto nitrocellulose membrane (Macherey&Nagel). Transferred proteins were detected by specific antibodies against D1 (Agrisera), Cytochrome f (Agrisera) and Rpl4 (Uniplastomic) and enhanced chemiluminescence reaction, ECL (Pierce).
BN-PAGE was conducted according to Schagger and von Jagow (1991 (link)) using the NativePAGE running buffer kit (Life Technologies). After BN-PAGE, the separated protein complexes were transferred to PVDF membrane (Macherey and Nagel) by western blot. Subsequently, the dye was removed from the membrane by incubation in methanol and the membrane was then used for immunodetection.

L. pneumophila JR32 was lysed by sonication (Bandelin, Sonopuls, Berlin, Germany) in a lysis buffer (150 mM sodium chloride, 1% Triton X-100, 50 mM Tris; pH 8.0), followed by Proteinase K treatment (0.05 mg/mL). Afterwards, the samples were pipetted onto a nitrocellulose membrane (Macherey-Nagel, Düren, Germany), and an immunoblot test was performed as described by Raulf et al. [46 (link)]. Briefly, the membrane was blocked with a 5% milk solution and then incubated with the respective CLR-Fc fusion proteins (1 µg/mL). For detection, an HRP-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Labs, RRID: AB_2337586) was diluted 1:10.000 in TBS + 0.05% Tween-20. The signals were visualized using Amersham ECL Prime Western Blotting Detection Reagent (Thermo Fisher, MA, USA). Chemiluminescence was measured using a chemiluminescence imager (Bio-Rad Laboratories, Hercules, CA, USA).

To quantify the amount of BVPrP produced at the different time points during the growth, 1 L culture was induced as described earlier and a 40 mL of sample was collected at 0, 1, 2, 4 and 16 h after induction.
Cells were collected by centrifugation at 15,000g for 10 min, weighted and resuspended in (0.1 g of cell paste/mL) volume of lysis buffer to normalize the cell content for each time point. For estimating the production of total prion protein, a 4 μL of lysis was mixed with 1 μL SDS loading buffer (5 ×) and boiled for 5 min prior to loading onto gels for Western blotting. To determine the amount of total soluble proteins expressed, resuspended cells were lysed by sonication and centrifuged at 18,000g for 20 min. A 4 μL of supernatant was mixed with 1 μL SDS loading buffer (5 ×) and boiled for 5 min prior to loading to gels for staining with Coomassie blue. Collected samples were analyzed on SDS-PAGE and by immunoblotting probed with monoclonal anti-His antibody (Sigma Aldrich) on nitrocellulose membranes (MACHEREY–NAGEL). The PrP bands were visualized by goat anti-mouse IgG, alkaline phosphatase conjugate (Sigma) using NBT/BCIP as substrate (Roche Diagnostics, GmbH). Intensity of the blot signals was quantified using the software LabImage 1D Gel Analysis (Kapelan GmbH, Germany).

Proteins were extracted using Tris-HCl buffer 0.1 M pH 7.5 that contained 1 mM EDTA, 150 mM NaCl, 0.1% NP40, 0.1 mM Na orthovanadate, 2 mM DTT, and 1 mM PMSF (lysis buffer), separated by SDS-PAGE, and transferred to nitrocellulose membranes (Macherey Nagel), which were then incubated with 5% non-fat milk in PBS-1% Tween. Primary antibody was added at the appropriate dilution and membranes were incubated for 16 h at 4°C. Primary antibodies used were: (1) rabbit polyclonal antibody anti-PKC-alpha (Cell Signaling Technology, 1/1000); (2) rabbit polyclonal antibody anti-Rac(1/2/3) (Cell Signaling Technology, 1/1000); (3) mouse monoclonal anti-c-Myc antibody (Santa Cruz Biotechnologies, 1/1000); (4) rabbit polyclonal antibody anti-HA-probe (Santa Cruz, 1/1000); (5) mouse monoclonal anti-alpha-actin (Millipore, 1/2000); (6) rabbit polyclonal antibody anti-LC3 (Sigma-Aldrich, 1/1000); (7) rabbit polyclonal antibody anti-GFP (Invitrogen, 1/1000); isotype-specific secondary antibody coupled with a horseradish peroxidase (Pierce, 1/2000) was detected by incubating with ECL+ (GE Healthcare) and visualized with CCD camera (FUJI Las 4000).

A total of 30 µg of muscle or cell extracts were loaded per well on a 10% acrylamide gel for SDS-PAGE. After migration, proteins were transferred onto nitrocellulose membranes (0.45 µm, Macherey Nagel), which were saturated 1 h with 5% non-fat milk in 0.5% Tween/PBS solution. Membranes were incubated with primary antibodies 1 h at room temperature or overnight at 4°C. Monoclonal mouse anti-vinculin (1/15,000, #V9264, Sigma-Aldrich), polyclonal rabbit anti-desmin (1/1,000, #C3956, Sigma-Aldrich), monoclonal rabbit anti-vimentin (1/2,500, #ab92547, abcam) and polyclonal rabbit anti-GAPDH (1/2,500, #G9545, Sigma-Aldrich) were used.
Isotype-specific anti-mouse or anti-rabbit secondary antibodies coupled with horseradish peroxidase (1/10,000, #31430 or #31460, Pierce, Thermo Scientific) were detected following incubation with Clarity Western ECL (BioRad) and visualized with a CCD camera (FUJI Las 4000 or Ai600, GE Healthcare).