Lung homogenates or cell lysates were subjected to denaturating SDS-PAGE, followed by electroblotting and immunoblotting for anti-ATF4, anti-GRP78, anti-CHOP (Santa Cruz Biotechnology, Dallas, TX), anti-ATF6α, anti-IRE1 (Enzo Life Sciences, Farmingdale, USA), anti-XBP-1 (Novus Biologicals, Littleton, CO), anti-eIF2α, anti-phospho eIF2α, anti-phospho PERK, anti-PERK, anti-phospho AKT(Thr308), anti-phospho p70S6K (Cell Signaling Technology, Danvers, MA) or anti-phospho IRE1 (Abcam, Cambridge, MA). Blots were developed using corresponding HRP-conjugated secondary antibodies and detected using a chemiluminescent system (Amersham ECL Plus; GE Healthcare, Piscataway, NJ). Band intensities were quantified with a LAS-1000 plus system (Fuji Film, Japan).
Las 1000 plus system
Manufactured by Fujifilm
Sourced in Japan
The LAS-1000 plus system is a laboratory equipment product by Fujifilm. It is a high-performance imaging system designed for scientific applications. The core function of the LAS-1000 plus system is to capture and analyze images of various samples, such as gels, membranes, and blots.
Automatically generated - may contain errors
Lab products found in correlation
Anti grp78, by Santa Cruz Biotechnology (1 mentions)
Anti phospho p70s6k, by Cell Signaling Technology (1 mentions)
Anti atf4, by Santa Cruz Biotechnology (1 mentions)
Anti phospho perk, by Cell Signaling Technology (1 mentions)
Anti chop, by Santa Cruz Biotechnology (1 mentions)
Anti phospho eif2α, by Cell Signaling Technology (1 mentions)
Amersham ecl plus, by GE Healthcare (1 mentions)
Anti xbp1, by Novus Biologicals (1 mentions)
Anti phospho ire1, by Abcam (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti phospho akt thr308, by Cell Signaling Technology (1 mentions)
Anti eif2α, by Cell Signaling Technology (1 mentions)
Chemiluminescence assay, by PerkinElmer (1 mentions)
Ab124954, by Abcam (1 mentions)
Ab40993, by Abcam (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Monoclonal anti flag m2 magnetic beads, by Merck Group (1 mentions)
Sypro ruby staining, by Bio-Rad (1 mentions)
Flag peptide, by Merck Group (1 mentions)
4 protocols using las 1000 plus system
1
Quantifying Cellular Stress Response Proteins
2
Western Blot Analysis of Protein Signaling
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Cell lysates were prepared with M-PER (Pierce, Rockford, IL, USA) plus protease inhibitor cocktail (HaltTM; Pierce) and protein concentrations were determined using the bicinchoninic acid assay (BCA) (Pierce). Aliquots of protein lysates were subjected to 10% SDS-PAGE, followed by electroblotting and immunoblotting for anti-p-AKT (Ser473) (1:1000, 3787S), anti-t-AKT (1:1000, 2920S), anti-a-caspase-3 (1:1000, 9661), anti-PARP (1:1000, 9542), anti-GAPDH (1:5000, 2118) (Cell Signaling Technology, Danvers, MA, USA), anti-t-caspase-3 (1:1000, ADI-AAP-113-D, Enzo), anti-actin (1:10000, 20536-1, Proteintech, Rocky Hill, NJ, USA), anti-GR (1:1000, E-AB-10354, Elabscience, Texas, USA), anti-GPX4 (1:1000, ab40993) and anti-TrxR (1:1000, ab124954, Abcam). Membranes were then probed with the indicated primary antibodies, followed by reacting with corresponding secondary antibodies, and detected using a chemiluminescence assay (PerkinElmer Life and Analytical Sciences, Boston, MA, USA). Membranes were exposed to X-ray film to visualize the bands (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Band intensities were quantified with a LAS-1000 plus system (Fuji Film, Japan).
3
Affinity Purification of Transgenic Mouse Brain Proteins
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Forty
whole brains without cerebellums from wild-type (negative control)
or BAC transgenic mice (ages from P14 to P17) were used for affinity
purification. The P2 fraction was cross-linked by DSP, and the membrane-bound
proteins were extracted as described previously. The cleared extraction
was incubated with monoclonal anti-FLAG-M2 magnetic beads (100 μL,
Sigma) at 4 °C overnight on a rotator. Anti-FLAG beads were pelleted
by a magnetic rack and washed five times with TBS containing 1% Triton
X-100. The bound proteins were eluted with 3×FLAG peptide according
to the manufacturer’s instruction (Sigma). The purified complexes
were resolved on a SDS-PAGE gel. 10% of the complexes were used for
immunoblot analysis, and 90% were used for SYPRO Ruby staining (Bio-Rad).
Protein bands were visualized using a UV light, and the image was
taken using an LAS-1000plus system (Fujifilm). The whole lane was
excised into 21 fractions and stored at −80 °C for further
mass spectrometry analysis.
whole brains without cerebellums from wild-type (negative control)
or BAC transgenic mice (ages from P14 to P17) were used for affinity
purification. The P2 fraction was cross-linked by DSP, and the membrane-bound
proteins were extracted as described previously. The cleared extraction
was incubated with monoclonal anti-FLAG-M2 magnetic beads (100 μL,
Sigma) at 4 °C overnight on a rotator. Anti-FLAG beads were pelleted
by a magnetic rack and washed five times with TBS containing 1% Triton
X-100. The bound proteins were eluted with 3×FLAG peptide according
to the manufacturer’s instruction (Sigma). The purified complexes
were resolved on a SDS-PAGE gel. 10% of the complexes were used for
immunoblot analysis, and 90% were used for SYPRO Ruby staining (Bio-Rad).
Protein bands were visualized using a UV light, and the image was
taken using an LAS-1000plus system (Fujifilm). The whole lane was
excised into 21 fractions and stored at −80 °C for further
mass spectrometry analysis.
4
Quantification of Smad1/5 Phosphorylation
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Cells were lysed with a lysis buffer [10 mMTris-HCl (pH 7.5) with 150 mM NaCl, 1%
Triton X-100, 0.1% SDS, complete protease inhibitor mixture (Roche, Basel, Switzerland), phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA)] and centrifuged at 15,000 rpm for 5 minutes at 4 ºC. Lysate proteins (20µg) were fractionated by 10% SDS-polyacrylamide gel electrophoresis, and separated proteins were transferred to a polyvinylidenedifluoride (PVDF) filter (Immobilon, Millipore, Billerica, MA, USA). The blots were blocked to reduce nonspecific signals with 1% bovine serum albumin in TBS-Tween. PVDF filters were probed with the following antibodies (Cell Signaling Technology, USA) shown in Table 1. Immune complexes were labeled with horseradish peroxidase-conjugated mouse or rabbit antibodies (BIOSOURCE International, Camarillo, CA, USA) and visualized by enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, NJ, USA) and a LAS-1000 Plus system (Fujifilm, Tokyo, Japan). To evaluate the difference of Smad1/5 phosphorylation status between the PS and PAMPS Cultures, the level of p-Smad1/5 was normalized by total Smad1 using image analysis software (NIH ImageJ 1.44p; National Institutes of Health, Bethesda, MD, USA).
Triton X-100, 0.1% SDS, complete protease inhibitor mixture (Roche, Basel, Switzerland), phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA)] and centrifuged at 15,000 rpm for 5 minutes at 4 ºC. Lysate proteins (20µg) were fractionated by 10% SDS-polyacrylamide gel electrophoresis, and separated proteins were transferred to a polyvinylidenedifluoride (PVDF) filter (Immobilon, Millipore, Billerica, MA, USA). The blots were blocked to reduce nonspecific signals with 1% bovine serum albumin in TBS-Tween. PVDF filters were probed with the following antibodies (Cell Signaling Technology, USA) shown in Table 1. Immune complexes were labeled with horseradish peroxidase-conjugated mouse or rabbit antibodies (BIOSOURCE International, Camarillo, CA, USA) and visualized by enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, NJ, USA) and a LAS-1000 Plus system (Fujifilm, Tokyo, Japan). To evaluate the difference of Smad1/5 phosphorylation status between the PS and PAMPS Cultures, the level of p-Smad1/5 was normalized by total Smad1 using image analysis software (NIH ImageJ 1.44p; National Institutes of Health, Bethesda, MD, USA).
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