Immunohistochemistry using anti-pSTAT3 (Cell Signaling, Danvers, MA) was carried out as previously described (Münzberg et al., 2003) with the following modifications: overnight fasted mice were treated by i.p. injection with 5 mg human recombinant leptin/kg body weight (R&D Systems, Abingdon, UK) 45 minutes before being perfused with 2% PFA, post-fixed overnight and processed to generate slides.
For preparing bacterial cell lysates, bacteria containing either pMMB67EHGm- pch::VSV-G or the vector alone were grown in identical conditions to those used for microscopy studies with the following modifications: overnight cultures were resuspended in 50 ml of fresh LB broth, and the fusion protein expression was induced by using IPTG at 250 μM at 0.1 OD600 nm.
To broaden the usefulness of ICSSWH systems, especially for overnight applications, numerous design modifications have been proposed and analyzed in the past.
CSF C3 and FH levels were measured using an xMAP technology-based multiplex human neurodegenerative kit (HNDG1-36 K; Millipore, Billercia, MA, USA) according to the manufacturer's overnight protocol with minor modifications.
Enumeration of bacteria from larval zebrafish was carried out as previously described with the modification that overnight growth was carried out at 28.5°C to recover commensal microbiota (Oehlers et al., 2011b).
Luminescence was detected according to the manufacturer's recommendations with minor modifications after incubation overnight at 4°C with the primary anti-RhoA antibody.
The labeled CN samples were then digested overnight using Glu-C, as modification of lysine residues by DMBNHS would result in missed cleavages at the Lys residue C-termini if trypsin or Lys-C was used.
Genomic DNA was obtained using the Raeder and Broda protocol ([1985]), with the following modifications: bacteria were grown overnight in 4 mL of DYGS medium (Rodrigues Neto et al. [1986]) and then centrifuged for 2 min at 10.000 g.
Using tandem mass-spectrometry, we could detect reversible oxidative modifications of C361 upon overnight incubation of Brf2 in absence of reducing agents and in presence of dimedone, a cyclic diketone that specifically reacts with sulfenic acid.
Assays were performed as described (Bertsche et al., 2006; Typas et al., 2010) with minor modifications: Cells were grown overnight, diluted 1 400 into 250 mL Lennox LB, and grown with shaking in baffled flasks at 30°C to OD600 = 0.3 0.4.
