Imagequant tl 1d

Protein (50 mg per sample) was loaded per line and separated on 8%–15% tris-glycine polyacrylamide gels and transferred to nitrocellulose membranes. Immunoblots were blocked for 2 h in Tris-buffered saline Tween-20 (TBST, 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, and 0.05% Tween 20) containing 5% skim milk. The blots were then incubated with primary antibodies in TBST at 4°C overnight. Membranes were washed three times with TBST and then incubated with secondary antibody for 2 h at room temperature. Membranes were washed again and developed with enhanced chemiluminescence (ECL) and detected with X-ray films. The blots were visualized with ImageQuant TL 1D (GE Healthcare, USA). Primary antibodies used from Cell Signaling Technologies (Danvers, MA, USA) were as follows: p-Tyr452 Gab2 (#3882, 1 : 1000), PI3 kinase p85 (#4257, 1 : 1000), Akt (#9272, 1 : 1000), p-Ser473Akt (#4058, 1 : 1000), p-Ser9GSK3β (#9323, 1 : 1000), and beta-actin (#3700T, 1 : 2000). Primary antibody directed against Grb2 (Abcam PLC, Cambridge, UK, #ab32111, 1 : 1000), GSK3β (Abcam PLC, Cambridge, UK, #ab131356, 1 : 1000), and Gab2 (Santa Cruz, USA, sc-365590, 1 : 500) were also used in the experiments. Secondary antibodies were goat anti-rabbit (BA1054, 1 : 2000) (Boster Biological Technology Co. Itd, Wuhan, China) or anti-mouse (BA1050, 1 : 2000).

The cells were harvested in peqGOLD TriFast reagent (VWR) and total RNA extraction was performed as recommended by the manufacturer’s protocol. 2.5 µg of total RNA were resolved on a 1% agarose/0.4 M formaldehyde gel using the tricine/triethanolamine buffer system as described^{15 (link)} followed by transfer on a nylon membrane (Roth) in 10x SSC. The blots were incubated overnight at 65 °C in Church buffer containing [α-³²P]-GTP body-labeled RNA probes for detection of the reporter and lacZ control RNA. Endogenous 7SL RNA was detected by a 5′-³²P-labeled oligonucleotide (5′-TGCTCCGTTTCCGACCTGGGCCGGTTCACCCCTCCTT-3′). The blots were visualized and quantified using the Typhoon FLA 7000 (GE Healthcare) and ImageQuant TL 1D software.

To administer an equal number of nucleocapsids of BVs and ODVs, we quantified the two different virions based on the relative amount of VP39. The quantification was performed by comparing the VP39 capsid mass through VP39 intensity in Western blot assays revealed by the monoclonal antibody to VP39. Images were digitalized and analyzed with Image Quant TL 1D software (GE, Healthcare). Additionally, polyhedra were counted in a Neubauer chamber and the content of VP39 was evaluated after dissolving polyhedra with 0.1 M sodium carbonate to release embedded ODVs.