2.1 Bacterial culture
Bacteria were streaked out and grown on Luria Broth (LB)-agar plates containing respective antibody for selection of transformants or in liquid culture. Transformed bacterial culture were maintained as glycerol stock, followed by freezing them in liquid nitrogen and subsequently stored at -700C.
2.2 Preparation of Ultracompetent cells
Ultracompetent cells were prepared using the Inoue method for preparation and transformation of E coli (Sambrook et al. 1987).
2.3 Plasmid Constructions
Ubx expression plasmid
Our lab had cloned the full length cDNA of Drosophila Melanogaster Ubx into pET 14b vector for protein expression. This fragment of Ubx having stop codon was PCR amplified from pET14b vector with PCR primers containing Kpn1 and BamH1 sites and was then sub-cloned into Kpn1 and BamH1 sites of a Drosophila expression vector, pRmHa-3GFP containing metallothionein promoter. Both the above constructs were sequence verified.
Forward primer: CCGGGTACCAGCGCAATGAACTCGTACTTTGA
Reverse primer: CGGGGATCCCTACTGATCTAAGTGTCCACCTTG
Reporter Plasmids
Addition of new motifs and control oligos into pDE5–37tkluc vector. Both sense and antisense oligos containing Kpn1 and Nhe1 were synthesized. Equal amounts of both sense and antisense oligos were heated for 10min at 99 0C and slowly allowed to cool. After that both oligo and Vector was digested with Kpn1 and Nhe1 restriction enzymes and was ligated with Promega fast DNA ligase. To identify positive clone of interest, Colony PCR was done using sense oligo as forward primer and RV primer1, RV primer3, RV primer 4 as reverse primer. Then they were sequence verified using GL primer 2 as forward primer.
Primer Used are:
RVprimer1 5’-CGACTCGAAATCCACATATCAAAT-3, RVprimer3 5´-CTAGCAAAATAGGCTGTCCC-3´
RVprimer4 5´-GACGATAGTCATGCCCCGCG-3´
For sequencing forward primer
GLprimer2 5´-CTTTATGTTTTTGGCGTCTTCCA-3´
2.4 Polymerase chain reaction (PCR)
After ligation and transformation positive colonies containing construct were identified using colony PCR. A master mix for PCR reaction was prepared on ice and randomly 5-10 clones were picked up from transformed master plate. The picked single colony was simultaneously being marked and maintained on other culture plate. Then same tips were kept inside PCR tubes. PCR reaction was set up using the specific primers along with a positive and a negative control. Then selected positive colony from colony PCR was further sequence verified.
2.5 Cell Culture
Drosophila S2 cells were maintained with standard methods. S2 cells were grown in Schneider’s media (invitrogen) supplemented with 10% fetal bovine serum, without any antibiotics. For transient transfection, 1.5 X 106cells per well in 6-well plates and 8-4.8X 108in 24 Well plates were seeded for experiments.
2.6 Extraction of plasmid DNA
Construct containing pDE5–37tkluc added oligos and pDE5–37tkluc as control were prepared using Sigma midi prep while other plasmids were prepared using maxiprep method (Sambrook et al. 1987) and were used for transfection.
2.7 Transient transfection and dual luciferase assay
S2 cells were grown in’s media (invitrogen) supplemented with 10% fetal bovine serum, without any antibiotics. Transient transfections into Drosophila S2 cells were carried out by effectene transfection reagent (Qiagen) as described by company. Total of 150ng of DNA was used for transfection in 12 well plates. Expression of Ubx protein in pRmHa3-Ubx transfected S2 cells were induced by 0.5mM copper sulphate after 18-20 h of transfection, cells were lysed after 24 hrs for extraction of protein and expression of proteins in cells were detected by immunofluoresence with N-terminal anti-Ubx antibodies as described below and by western blot analysis.
For all reporter assay Co-transfections into Drosophila S2 cells were carried out by effectene transfection reagent (Qiagen). Expression vector to reporter was 1:5 or 1:1. Expression of Ubx protein in pRmHa3-Ubx transfected S2 cells were induced by 0.5mM copper sulphate after 18-20 h of transfection, cells were lysed after 24 hrs of induction. The luciferase reporter activity was determined with the dual luciferase reporter assay system (Promega).
2.8 Antibody Staining for S2 cells
Preparation of Cover slip: Before growing cells on cover slip they were soaked in 1N HCl for overnight at 600C in oven. Then were washed thoroughly with autoclaved water and were stored in pure ethanol.
After that cover slips were dried in a large plastic Petri dish without filter paper in Tissue culture hood. They were kept such that they do not touch each other. 0.5 mg/ml solution of ConA filter sterilized (Sigma) in water was added with pipette and was kept for 30min-1hr. ConA on cover slip was washed with PBS and was air dried.
For detecting protein of interest cells were cultured on glass cover slips coated with conA. Then expression plasmids were transformed. For staining cell culture media was removed and cells were fixed with 3.5% paraformaldehyde and 0.05% glutraldehyde in PHEM buffer saline (60mM PIPES, 25mM HEPES, 10mM EGTA, 2mM MgCl2, 6H2O) for 10 min. Cells were washed 3 times with PHEM buffer saline and were permeabilized with 0.1% Triton X
100 in PHEM for 10 min. Cells were blocked with 5% heat inactivated goat serum in PHEM buffer for one hour. The cells were incubated with primary polyclonal Nterminal anti-Ubx (1:1000) overnight at 4 °C. After that 4 washes for 10 min with PHEM buffer was given and fluorescein secondary antibodies (1:1000) were applied for one hour at room temperature in dark. Final 3 washes for 10 min were performed. Cover slip with antifade reagent with DAP1 was placed gently on slide to visualize the protein by fluorescence microscopy.
2.9 Western blot analysis
Cultured suspension S2 cells were centrifuged at 450Xg for 5min to pellet cells and media was removed. Cells were given wash with PBS and were further centrifuged to remove PBS. RIPA lysis buffer or passive lysis buffer was added and was mixed by vortexing. Kept on ice for 15 min and further votexed briefly to re-suspend cells and lyse residual cells. Whole adult and larvae samples were frozen in liquid nitrogen after that were crushed and lysed in RIPA lysis buffer. Samples were quickly frozen in liquid nitrogen and stored at -700C for future use. Then stored samples were centrifuged to remove cell debris and supernatant was treated for loading on gels. Protein harvested from cell was further boiled for 5min in Laemmli’s buffer and separated on 10% SDS-PAGE (Sambrook et al. 1987).
The resolved gel was blotted on PVDF membrane and was stained with Ponceau S to check the protein. The membrane was blocked with 5% skimmed milk for 1X TBST Membrane was given three wash for 10min at RT with 1X TBST. The transblot was probed with antibodies against polyclonal N-terminal Ubx antibody (Agrawal et al., 2011) diluted to 1:5000 and followed by secondary horseradish peroxidase-conjugated antibodies for 1h at RT or overnight at 4°C.