Hybond c extra

Cell extracts were separated under reducing electrophoresis on a 1mm of NuPAGE Novex Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane (HybondTMC extra, Amersham Biosciences). Tuj1, Actin and cleaved caspase-3 proteins were stained with α-Tuj1 (Sigma-Aldrich, T8660), α-cleaved caspase-3 (Cell Signaling, #9664) and α-actin (Sigma-Aldrich, A4700) at 1/1000 dilution for 2h at room temperature. Blots were developed using the ECL (enhanced chemiluminescence) detection system after incubation with HRP-labeled anti-mouse IgG anti- body (Amersham Biosciences Bioscience), 1/5000, 1h of room temperature incubation.

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail (Roche) (52 (link)). Proteins were separated by SDS-PAGE before transfer onto nitrocellulose membrane (Hybond C Extra; Amersham Biosciences). Membranes were probed with the appropriate primary and horseradish peroxidase (HRP)-conjugated secondary antibodies. Proteins were detected using EZ-ECL enhancer solution (Geneflow) as previously described (53 (link)).

Western blots were performed as previously described [56 (link)]. Nine micrograms of proteins of control and ES cells (500 mV–100 Hz) were loaded on a 12% polyacrylamide gel for electrophoresis (SDS–PAGE; Bio-Rad) and transferred to a nitrocellulose membrane (Hybond-C Extra; Amersham, Barcelona, Spain). Membranes were blocked for 1 h, and finally incubated overnight at 4 °C, with the following primary antibodies: (i) rabbit polyclonal PathScan® Multiplex Western Cocktail antibodies against Phospho-p90RSK, Phospho-Akt, Phospho-p44/42 MAPK (Erk1/2) and Phospho-S6 Ribosomal (#5301 Cell Signaling, Danvers, MA, USA); (ii) rabbit polyclonal antibody against Phospho-GSK-3β (Ser9) (#9336 Cell Signaling, Danvers); and (iii) mouse monoclonal antibody against β-actin (A1978 Merck Life Science S.L.U. Madrid, Spain). After that, membranes were incubated with the appropriate anti-rabbit or anti-mouse secondary antibody (Dako) horseradish peroxidase-conjugated, at a dilution of 1/5000 and developed using the ECL-plus detection method (Amersham) and the ImageQuant LAS 4000 MINI GOLD (GE Healthcare Life Sciences, Barcelona, Spain). For quantification, the optical density of individual bands was analyzed using the ImageJ software (NIH, USA), and the optical density of each band was normalized relative to the optical density of β-actin.

For Western blot analysis, 25 to 60 µg of proteins from gels was electrotransferred to Hybond C Extra membrane (Amersham Biosciences). The blots were processed as described by Rao et al. (25 (link)).

Protein-protein interactions between PilG_FL, partial proteins and endoproteinase cleavage products, in addition to other pilus biogenesis proteins, were assessed by a solid phase protein overlay assay as previously described [11 (link), 17 (link)]. Briefly, 1 μg of purified recombinant PilG, PilQ [55 (link)], PilN, PilO, PilP [11 (link)], PilF, PilT, ComP, ComL proteins, purified pili [59 ] and BSA were applied to SDS-PAGE and transferred onto nitrocellulose membranes (Hybond-C Extra, Amersham Biosciences) in Towbin transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% methanol, 0.1% SDS, pH 8.3). The membranes were briefly washed twice with renaturing buffer (0.25% gelatin, 0.5% BSA, 0.2% Triton X-100, 10 mM Tris-HCl, 5 mM β-mercaptoethanol, 100 mM NaCl, pH 7.5), and the proteins were renatured by incubation at 4°C overnight in the same buffer. For the detection of protein-protein interaction, the membranes were incubated for 3 h with 1 μg purified full-length PilG or PilQ or with partial proteins thereof in 10 ml renaturing buffer, and washed in a Tris-buffered saline (100 mM Tris-HCl, 150 mM NaCl, pH 7.5). Bound PilG or PilQ were detected with specific rabbit antisera. The PilQ-PilP interaction was used as a positive control [11 (link)], while BSA was used as a negative control. All the experiments were repeated at least three times.

Western blotting was performed as previously described (Davidson et al., 2013b (link)). In brief, equal numbers of cells were pelleted and immediately lysed with 70°C 1× NuPAGE LDS sample buffer (Life Technologies) containing 50 mM DTT reducing agent. The lysates were then separated using 10% Bis/Tris NuPAGE polyacrylamide gels (Life Technologies). After SDS-PAGE, proteins were transferred onto nitrocellulose membranes (Hybond-C-extra; Amersham Biosciences) in a BioRad transfer tank filled with 1× SDS-transfer buffer at 100 V for 1 h. The membranes were then blocked with a solution containing 5% nonfat dried skimmed milk dissolved in TBS, after which they were probed with primary antibodies (1:1,000 dilution) overnight at 4°C. Detection was achieved through the use of HRP or fluorescently conjugated secondary antibodies combined with chemiluminescence (Immobilon Western chemiluminescent HRP substrate; Millipore) or an Odyssey IR imaging system (LICOR Biosciences). Coomassie staining or probing for MCCC1 with Alexa Fluor 680–conjugated streptavidin (Davidson et al., 2013a (link)) was used to confirm equal loading.

Cells were lysed in a solution containing 50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA and a cocktail of protease inhibitors^{48 (link)}. Proteins were separated in 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, and transferred to nitro-cellulose membrane (hybond C-Extra, Amersham Biosciences). After blocking with 5% non-fat milk in PBS-0.05% Tween, membranes were incubated with primary and HRP-secondary antibodies and the signals detected by Chemiluminescent substrate HRP (Thermofisher, Millipore) using a conventional developer or a digital detection system (ChemiDoc, Biorad). Alternatively, fluorescent-labelled secondary antibodies were used and detection was performed thanks to the Odyssey® CLx imaging system (LI-COR). Uncropped scans of immunoblots are included as Supplementary Fig. 5. For immunofluorescence, cells were fixed with 4% Paraformaldehyde (PFA) for 15 min Blocking and permeabilisation were performed in PBS/0.1%Triton, 4% Bovine Serum Albumine (Sigma), 4% Donkey Serum (Sigma) for 1 h at room temperature. Subsequently primary antibodies, diluted in blocking buffer was incubated overnight at 4 °C. Following washes with PBS/0.1%Triton, secondary antibodies was incubated for 1 h at room temperature, and mounted in Mowiol solution (Sigma, 81381).