Chemiluminescence kit

Anti-GFAP and anti-SOX2 antibodies were obtained from Dako, Agilent (Santa Clara, CA, United States), anti- VDR and anti-aSMase were from Elabscience (Houston, TX, United States) and anti-β tubulin was from Sigma Aldrich (St. Louis, MO, USA). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TaqMan SNP Genotyping Assay and Reverse Transcription kit were purchased from Applied Biosystems (Foster City, CA, USA). RNAqueous^®-4PCR kit was from Ambion Inc. (Austin, Texas). SDS-PAGE molecular weight standards were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Chemiluminescence kit was purchased from Amersham (Rainham, Essex, UK).

Cell lysates and tissue homogenates were prepared by using chilled RIPA cell lysis buffer (containing protease inhibitors 50 × 20 μL) with gentle agitation. After high-speed hypothermal centrifugation, the supernatant was collected and the total protein concentration was determined by BCA kit. Supernatant protein samples were separated by 12.5% SDS-PAGE and electro-transferred to PVDF membranes. Subsequently, the membranes were sequentially blocked by TBST (containing 5% skim milk), and co-incubated by primary and secondary antibodies. Representative immunoblottings of protein were visualized by a chemiluminescence kit (Amersham Biosciences, USA).

Polarized macrophages were stimulated with poly (I:C) as mentioned above. Later, cells were harvested, washed, and lysed in lysis buffer (RIPA buffer, protease, and phosphatase inhibitor cocktail). In SNs, proteins were estimated and equal concentration was subjected to SDS-PAGE. After transfer to nitrocellulose membrane and subsequent blocking, the membranes were immunoblotted with Abs against iNOs and actin as a loading control. Blots were developed using chemiluminescence kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Blots were scanned with the help of phosphoimager (Fujifilm, Tokyo, Japan), and image analysis was performed with MultiGuage software.

For iNOs, nDCs harvested on 6 d of culture were stimulated with LPS for 24 h or infected with Mtb for 4 h. Mtb infected DCs were washed to remove extracellular bacteria and cultured for 24 h. After 24 h, cells were washed, and lysed in lysis buffer (RIPA buffer, protease and phosphatase inhibitor cocktail). In SNs, proteins were estimated and equal concentration was subjected to SDS-PAGE. After transfer to nitrocellulose membrane and subsequent blocking, the membranes were immunoblotted with Abs against iNOs and actin (loading control). Blots were developed using chemiluminescence kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Blots were scanned using phosphoimager (Fujifilm, Tokyo, Japan) and images analyzed employing MultiGuage software. pStat-1, pStat-4 and p38 were detected in the lysate of nDCs harvested on 6 d of culture following the same protocol as mentioned above. The fold change in the phosphorylation of Stat-1, Stat-4 and p38 was calculated as a ration between phosphorylated and un-phosphorylated forms.

Amino acid substitution at T143A in H7N7HA wild-type protein inhibits synthesis of N- glycosylation motif in A141. It was confirmed by western blot analysis. Briefly, the Bac-HA or Bac-HA mutant infected cell supernatant was mixed with Laemmli sample buffer and resolved in 12% SDS–PAGE. The gel was transferred to a nitrocellulose membrane and Western blotting was performed as described previously Kumar et al (2013)[8 (link)].The anti-mouse rHA polyclonal antibody (TLL, Singapore) at a dilution of 1:250 was used as the primary antibody and rabbit anti-mouse Ig (Dako Cytomation, Denmark) at a dilution of 1:3000 was used as a secondary antibody. The protein bands were visualized by chemiluminescence kit (Amersham, UK).

RNA isolation and quantitative real-time PCR (qPCR) were performed using the standard curve method as previously described [33 (link),34 (link)]. The probes, Ace1 (Rn00561094_m1), Ace2 (Rn01416289_m1), Tmprss2 (transmembrane protease serine 2, Rn00590459_m1) and 18sRNA (Hs99999901_s1, endogen reference) were purchased from Life technologies (Darmstadt, Germany) and span exon-exon boundaries. Membrane proteins of the duodenum, kidneys, heart and lungs (n = 5/per group) were isolated using MembraneMax™ Protein Expression Kits (ThermoFisher Scientific, Germany). Proteins were detected by incubating with HRP-conjugated secondary antibodies at a 1:3000 dilution (Dianova, Hamburg, Germany) at room temperature for 2 h and a chemiluminescence kit (Amersham Pharmacia Biotech, Freiburg, Germany). Equal protein loading was verified using mouse anti-ß-actin (2 μg/mL; #3700; Cell Signaling Technology). ACE2 serum concentrations were measured by ELISA using rat standards according to the manufacturer’s protocol (Angiotensin I Converting Enzyme 2 ELISA; Cloud-Clone Corp., #SEB886Ra, Houston, TX). Circulating HbA1c percent levels were measured using the Hitado Super ID (Möhnesee, Germany). For analysis SigmaStat (Jandel Scientific, San Rafael, CA, USA) was used.