1d image software

For lysate preparation, cells were lysed as described in [15 (link)]. Protein concentration was determined by the Bradford method. Equal amounts of proteins were loaded on 11% SDS-PAGE, blotted on Immobilon-P membranes (Millipore), and processed in Western blot (WB) with the indicated antibody, detected by chemiluminescence. Quantitation of the signal was obtained by chemiluminescence detection on a Kodak Image Station 440MM PRO and analysis with the Kodak 1D Image software.

Whole protein, including Occludin and TJP-1, which are responsible for maintaining the intestinal barrier function [72 (link)], and TLR4 and NF-κB signaling pathway, which are highly crucial for modulating inflammatory responses [52 (link)], from the liver and cecum mucous were lysed by RIPA lysis buffer (LifeTechnologies Inc., USA) supplemented with protease inhibitor cocktail (Roche, USA). The protein concentration in the tissue lysate was measured with BCA. Proteins were loaded onto the SDS-PAGE gel (BioRad) and electrophoresed and analyzed by WB using antibodies against TLR4 (BA1717, Boster, Wuhan, China), MYD88 (abs135682, Absin, Shanghai, China), IKBα (D120138, Sangon Biotech, Shanghai, China), p-IKBα (D151548, Sangon Biotech, Shanghai, China), p-p65 (HZ4902812, TW reagent, Shanghai, China), TJP-1 (mAb13663, Cell Signaling, USA), Occludin (ab167161, Abcam, USA), and actin (60008, Proteintech, USA). The bands were detected using the chemiluminescence kits (Amersham Biosciences, UK). Chemiluminescence was recorded with an Image Station 440CF, and results were analyzed with the 1DImage Software (Kodak Digital Science, Rochester, NY, USA).

Cell extracts were obtained as described by Beretti et al. [21 (link)]. Briefly, subconfluent cells were extracted by the addition of AT lysis buffer (20 mM Tris-Cl, pH 7.0; 1% Nonidet P-40; 150 mM NaCl; 10% glycerol; 10 mM EDTA; 20 mM NaF; 5 mM sodium pyrophosphate; and 1 mM Na₃VO₄) and freshly added protease inhibitor cocktail at 4°C for 30 min. Lysates were sonicated, cleared by centrifugation, immediately boiled in SDS sample buffer, and centrifuged. Supernatants were loaded onto SDS-polyacrylamide gel, blotted on Immobilon-P membranes, and processed by western blot with the indicated antibodies, detected by SuperSignal substrate chemiluminescence detection kit. Quantitation of the signal was obtained by chemiluminescence detection on a Kodak Image Station 440CF and analysis with the Kodak 1D Image software. Primary antibodies were raised against the following molecules: rabbit-p16 and rabbit-β-actin.

Protein lysates were prepared from mouse tissue or C2C12 cells using lysis buffer containing 0.3% Triton X-. Protein content was determined by the BCA protein assay (Pierce, Rockford, IL). Total protein (50 μg) was separated by SDS-PAGE [10% (w/v)] and transferred to PVDF membranes (Bio-Rad, Hercules, CA). Membranes were first blocked for 1 hat 25°C in 4% (w/v) instant milk dissolved in 0.1% Tris-buffered saline with Tween 20 TBS (TTBS) and incubated with the following primary rabbit-raised antibodies (1:1,000 dilution in TTBS): Myostatin (19142-1-AP, Proteintech; RRID:AB_10638615), AMPD1 (19780-1-AP; RRID:AB_10644281), GLUL (80,636, Cell Signaling; RRID:AB_2799956), pIRS1^Ser636/639 (2388, Cell Signaling; RRID:AB_330339), IRS1 (2382, Cell Signaling; RRID:AB_330333), pAKT^Ser473 (4058 Cell Signaling; RRID:AB_331168), AKT (9272, Cell signaling; RRID:AB_32982) and GAPDH (5174, Cell Signaling; RRID:AB_10622025) and visualized using an anti-rabbit (no. 7074) horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000, Cell Signaling) using the HRP Immunstar detection kit (Bio-Rad). Chemiluminescence was recorded with an Image Station 440CF, and results were analyzed with the 1D Image software (Kodak Digital Science, Rochester, NY). Data for proteins of interest are expressed normalized to GAPDH expression.

Protein lysates were prepared from mouse tissue employing MAP Kinase lysis buffer as previously described (47 (link)). Protein content was determined by the BCA protein assay (Pierce). Total protein (50 μg) was separated by SDS-PAGE (10% w/v) and transferred to PVDF membranes (BioRad). Membranes were first blocked for 1 hour at 25°C in 4% (w/v) instant milk dissolved in 0.1% Tween-20 Tris-Buffered Saline (TTBS); incubated with primary rabbit or mouse-raised antibodies (1:1000 dilution in TTBS) KHK (Sigma, HPA007040; RRID: AB_1079185), FAS (Cell Signaling, 3180; RRID: AB_2100796), ACC (Cell Signaling, 3676; RRID: AB_2219397), V1bR (BIOSS; bs-11800R), and actin (Cell Signaling, 4968; RRID: 2313904); and visualized using an anti-rabbit (7074; RRID: AB_2099233) or anti-mouse IgG (7076; RRID: AB_330924) horseradish peroxidase–conjugated secondary antibody (1:2000, Cell Signaling) using the HRP Immun-Star Detection Kit (Bio-Rad). Chemiluminescence was recorded with an Image Station 440CF and results analyzed with the 1D Image Software (Kodak Digital Science). See complete unedited blots in the supplemental material.

Equal amounts of cellular proteins (10 μg) were loaded on 11% SDS/PAGE, blotted on Immobilon-P membranes (Millipore), processed by western blot with the indicated antibodies, and detected by chemiluminescence on a Kodak Image Station 440MM PRO. Quantitation of the signal was obtained by analysis with the Kodak 1D Image software.
Akt immunoprecipitations were performed from 50 μg of total protein lysates, from HA-tagged Akt isoforms transfected cells, in 30 μl of final volume. Lysates were incubated overnight at 4°C with 1.3 μl of anti-HA antibody followed by 25 μl protein A-Sepharose (Sigma-Aldrich) for 40 min at 4°C. Immunocomplexes were washed twice with Tris/HCl 50 mM (pH 7.5), once with Tris/HCl 5 mM (pH 7.5) and used as CK2 substrate in in vitro phosphorylation assays.