Ab6556

MEFs were seeded on coverslips and prepared as described previously (Srivastava et al., 2015 (link)). To induce cellular stress, 1 mM arsenite was added to cultures for 1 hr. Stains with primary antibodies were carried out using optimized conditions overnight at 4°C in a humid chamber. The primary antibodies were goat anit-AMPKα1, clone C20 (Santa Cruz) at 1:75 ; with either rabbit anti-ROQUIN at 1:75 (Cat. NB100-655, Novus Biologicals) or rabbit anti-GFP 1:1000 (Cat. ab6556, Abcam, UK). Images were taken on a Leica SP5 confocal microscope with a pin hole of 67.9 μm and an APO CS 1.25 UV x40 oil objective. Higher magnification images presented in Figure 3c were taken on a Leica SP5 confocal microscope with a pin-hole of 95.5 μm and an HCxPL APO lambda blue x63/1.4 oil objective.

For sporulation time-course experiment, cells were harvested at different time points during sporulation and protein was isolated by trichloroacetic acid (TCA) precipitation method. Protein samples were electrophoretically resolved on SDS-polyacrylamide gels and subjected to Western blot analysis. Rabbit polyclonal anti-Protein A antibody (P3775) (1:1,000) from Sigma was used to detect Sub1-TAP protein. Mouse monoclonal anti-beta Actin antibody (ab8224) (1:2,500) from Abcam was used for loading control. For GFP expression in mitotically growing cultures, rabbit polyclonal anti-GFP antibody (ab6556) (1:1,000) from Abcam was used. HRP conjugated goat anti-rabbit or anti-mouse (1:10,000) from Sigma were used as the secondary antibodies. Quantification of the protein bands was carried out using multi-gauge software.

The anti-GAUT12 polyclonal antibody was generated against synthetic peptides corresponding to GAUT12 amino acid residues 101–114 (EQPLSEQELKGRSD) and antigen-purified over a column packed with the antigenic-peptide (service via New England Peptide, http://www.newenglandpeptide.com/). The purified anti-GAUT12 antibody did not cross-react with GAUT1 or GAUT7 (Supplemental Figure S11). Pre-immune serum did not contain anti-GAUT12 antibody. Anti-GAUT1 and anti-GAUT7 antibodies were generated previously (Sterling et al., 2006 (link); Atmodjo et al., 2011 (link)). For western blotting, a dilution factor of 1:5000, 1:10000, 1:3000, and 1:2000 was applied for anti-GAUT12, anti-GAUT7, anti-GAUT1, and anti-GFP (abcam, ab6556), respectively. Either horseradish peroxidase (HRP)- or alkaline phosphate (AP)-conjugated goat anti-rabbit secondary antibody (Sigma) was used followed by a reaction with corresponding substrates to yield a blue or purple color precipitant on the target protein band.

First, ~750 synchronized late L4 to young adult stage animals were plated on NGM plates seeded with EHEC strain EDL933 or control plates seeded with E. coli OP50 for 4 days at 20 °C. Polyclonal antibody to GFP (Abcam, ab6556), monoclonal antibody to SUN-1 (phospho-S43) (University of Vienna, Austria), and monoclonal antibody to α-tubulin (Sigma-Aldrich, T6199) were used for detection, Caco-2. In brief, 3 × 10⁵ cells were seeded to a 10-cm cell culture dish. When monolayer cells became 70–80% confluent 4–6 days after the inoculum, they were infected by EHEC strain EDL933 at MOI (500:1) for 0.5 h. Monoclonal antibody to GAPDH (Abcam, ab181602), polyclonal antibody to histone H3 (phospho-S10) (Abcam, ab47297), monoclonal antibody to DIAPH1 (Cell Signaling, 14634), polyclonal antibody to DIAPH2 (Cell Signaling, 5474), polyclonal antibody to DIAPH3 (Sigma-Aldrich, SAB1409850), and monoclonal antibody to polyclonal phospho-PP1α (Thr320) (Cell Signaling, 2581) were used for detection.

Prior to immunolabeling, grids were placed on plates with solidified 2% gelatine and warmed up to 37°C for 20 min to remove the pick-up solution. After quenching of free aldehyde-groups with glycine (0.1% for 15 min), a blocking step with 1% BSA (fraction V) in 0.1 M Sörensen phosphate buffer (pH 7.4) was performed for 40 min. The grids were incubated in primary antibody, rabbit polyclonal to GFP (ab6556, Abcam, UK), diluted 1:125 in 0.1 M Sörensen phosphate buffer over night at 4°C, followed by a 2 hr incubation in the secondary antibody, a goat-anti-rabbit antibody coupled with 6 nm gold (GAR 6 nm, Aurion, The Netherlands), diluted 1:20 in 0.1 M Sörensen phosphate buffer, performed at RT. The sections were stained with 4% uranyl acetate (Merck, Germany) and 2% methylcellulose at a ratio of 1:9 (on ice). All labeling steps were conducted in a wet chamber. The sections were inspected using a FEI Morgagni 268D TEM (FEI, The Netherlands) operated at 80kV. Electron micrographs were acquired using an 11-megapixel Morada CCD camera from Olympus-SIS (Germany).

BN-PAGE gels were destained with 40% (v/v) methanol, 10% (v/v) glacial acetic acid for 30 min, refreshing the destaining solution every 10 min. Gels were soaked for 30 min in 1% SDS in Tris-buffered saline, pH 7.4, at room temperature. Proteins were blotted onto PVDF membrane by wet transfer at 100 V for 1 h. Coomassie-stained BSA marker bands were marked onto the membrane using a felt-tipped pen. Membranes were blocked in 20% (w/v) Marvel-skimmed milk powder in 0.1% PBS-Tween for 1 h. Membranes were incubated overnight at 4 °C on a roller in rabbit anti-AQP4 antibody (Abcam, ab128906) diluted 1:5,000 or rabbit anti-GFP (Abcam, ab6556) diluted 1:10,000, both in 5 ml of 0.1% PBS-Tween. Membranes were washed in 0.1% PBS-Tween and incubated with donkey anti-rabbit HRP (Santa Cruz, sc-2313) diluted 1:10,000 in 20 ml of 0.1% PBS-Tween at room temperature for 1 h. HRP was detected on x-ray film using ECL reagent (Amersham Biosciences).

Protein samples were separated on 10 % acrylamide gels, and subjected to western blotting on polyvinylidene difluoride membrane (Immobilon-P; Millipore). Blots were blocked with 5 % skim milk in phosphate-buffered saline with Tween (PBST; 7 mM Na₂HPO₄, 3 mM NaH₂PO₄, 140 mM NaCl, 5 mM KCl, 0.05 % Tween-20) for 1 h at room temperature. For detection of GFP, blots were incubated with rabbit polyclonal anti-GFP (Abcam ab6556, 1:1000) or anti-biotin (Abcam ab1227, 1:1000) in PBST + 5 % skim milk for 1 h at room temperature, washed with PBST three times for 10 min each at room temperature, incubated with anti-rabbit IgG antibody conjugated to horseradish peroxidase (Jackson Immuno Research 111035003, 1:10,000) for 45 min at room temperature, washed with PBST three times for 10 min each at room temperature, and finally washed once with PBS at room temperature for 10 min. Blots were developed using enhanced chemiluminescent western blotting substrate (Bio-Rad Laboratories).