Image multi gauge software

The cells were lysed and immunoblotted as previously described [43 (link)]. Briefly, proteins were resolved by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF membrane (Millipore, Billerica, MA, USA) and were detected by the proper primary and secondary antibodies before visualization with a chemiluminescence kit (Biological Industries, Kibbutz Beth HaEmek, Israel). Visualization was performed with Image Quant LAS-4000 (Fujifilm, Tokyo, Japan) using image Multi-Gauge Software (Fujifilm).

Frozen mouse liver tissue samples (about 50 mg) were homogenized in 650 mL of TNE buffer (1 mmol/L EDTA, 1% Nonidet P40, 10 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl) containing a phosphatase inhibitors and cocktail of protease (Roche, Basel, Switzerland). The incubation condition was 4°C for 45 min and the centrifugation condition was at 12 000 g for 5 min. BCA Protein Assay (Thermo Fisher, MA, USA) was conducted to quantified protein content was quantified in the supernatant. Reducing sample buffer (Bio-Rad) was used to mix lysates for electrophoresis and subsequently the lysates were transferred onto polyvinylidene fluoride membranes (Thermo Fisher). Equal loading (50 mg) was verified using Ponceau red solution. Antibodies against TSLP (ab188766) was purchased from Abcam (MA, USA) and p-JAK1 (#3331S), JAK1 (#50994S), p-STAT1 (#9167S), STAT1 (#14994S), p-STAT3 (#52075S), STAT3 (#9139S) and anti-β-actin (#3700S) were obtained from Cell Signaling Technology (Danvers, MA, USA). After incubating with HRP-labeled goat anti-rabbit IgG (H+L) (1:3000; Beyotime, Shanghai, China), the Clarity Western ECl Substrate (BioRad, CA, USA) was conducted to proceed immunodetection and the Las-4000 Imaging System and Image MultiGauge software (Fujifilm, Tokyo, Japan) were used to reveal bands.

The experiment was performed essentially as described before (Nordfelth and Wolf-Watz, 2001 (link)). In short, 5 × 10⁶ HeLa cells were infected with induced Yersinia strains at a MOI of 50:1 in the presence of Cytochalasin D (0.5 μg/ml). After infection the cells were washed in PBS and treated with 0.5 mg/ml Proteinase K (Roche Diagnostics, Gmbh) for 1 min. The cells were aspirated and incubated 20 min at room temperature, after which, 4 mM PMSF was added to inactivate remaining Proteinase K. The cells were lysed with 0.4% Digitonin in a total volume of 1 ml. Cell debris and bacteria were pelleted by centrifugation at 15,000 g, 10 min, 4°C. The supernatant was separated on a 12% SDS-PAGE and analyzed by Western blot using affinity purified antisera against YopE and YopH or monoclonal Tubulin antibody (Sigma Aldrich). The protein levels were quantified by use of the LAS4000 image reader and Multi Gauge-Image software (Fujifilm). The levels of YopE and YopH were normalized against Tubulin levels. The results were analyzed using the paired Student's t-test with the significance set at p ≤ 0.05^*, p ≤ 0.01^** and p ≤ 0.001^***.

Digital autoradiography of ¹²⁴I-H12(CAT) on the A431 xenograft was performed ex vivo following the whole body PET-CT analysis. Tumor sections were placed on a phospholuminescence plate for approximately ten minutes. The plate was scanned in a Bio-Imaging Analyser BAS-1800II (FujiFilm Medical Systems), and the digital images were processed using Multi Gauge Image software (FujiFilm Medical Systems).