To extract protein, liver samples were homogenized using a Polytron PT2100 homogenizer in cell lysis buffer (Cell Signaling, Danvers, MA), containing phosphatase and protease inhibitors, and quantified using Bradford reagent (Sigma-Aldrich, St. Louis, MO). Mitochondrial fractions were prepared as described previously [20 (link)]. Equal amount of protein (40 μg) from each sample was resolved on SDS-PAGE, trans-blotted onto a PVDF membrane (Bio-Rad, USA) and subjected to immunoblot assay by using primary antibodies (1:1000) for JNK, Bax and p-JNK (Cell Signaling, Danvers, MA) followed by secondary anti-rabbit horseradish peroxidase antibody (1:5000; Cell Signaling, Danvers, MA). Blots were developed with chemiluminescence reagent (Bio-Rad, Hercules, CA) using autoradiography films (Genesee Scientific, San Diego, CA). Blots were stripped using stripping buffer (Thermo Scientific, Wilmington, DE) and re-probed with goat anti-rabbit β-actin antibody (1:5000; Cell Signaling, USA) to determine equivalent loading. Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/ ).
Autoradiography film
Manufactured by Genesee Scientific
Sourced in United States
Autoradiography film is a type of photographic film used to detect and visualize radioactive signals, such as those emitted from labeled molecules or cells in biological experiments. It functions as a recording medium for the detection and analysis of radioactive samples.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence reagent, by Bio-Rad (3 mentions)
Coomassie protein assay reagent, by Merck Group (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (2 mentions)
Ripa cell lysis buffer, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Improm 2 reverse transcription system, by Promega (2 mentions)
Ecl reagent, by Bio-Rad (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Cfx384 touch real time pcr detection system, by Bio-Rad (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Stripping buffer, by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Goat anti rabbit β actin antibody, by Cell Signaling Technology (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
P21waf cip1, by Cell Signaling Technology (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
P16ink4a, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse and anti rabbit antibodies, by Cell Signaling Technology (1 mentions)
11 protocols using autoradiography film
1
Liver Protein Extraction and Immunoblotting
2
Age-related RPE Protein Expression
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Protein was extracted from RPE/eyecup of mice (2, 12 and 18 months old) or ARPE-19 cells or primary RPE cells using RIPA cell lysis buffer (Thermo Scientific) containing protease and phosphatase inhibitors and concentration was determined using the coomassie protein assay reagent (Sigma-Aldrich, USA). Equivalent amount of protein samples were subjected to SDS–PAGE, transferred to PVDF membranes, and then incubated with primary antibodies: SIRT-1, p21Waf/Cip1 (1:1000; Cell signaling) and p16INK4a (1:500; Abcam) overnight at 4°C. Next day, blots were washed with TBST and incubated with horseradish peroxidase conjugated secondary antibody (1:3000; Sigma-Aldrich, USA) for 60 min with gentle shaking at room temperature. Blots were then washed (with TBST) and developed with chemiluminescence reagent (Bio-Rad, Hercules, CA) using autoradiography films (Genesee Scientific, San Diego, CA). β-actin (1:3000; Sigma-Aldrich, USA) expression was evaluated to determine equivalent loading. Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/ ).
3
Western Blot Analysis of Redox Regulators in Mouse and Human RPE
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Total protein was extracted from RPE/eyecup of mice or from human RPE (ARPE-19) cells using RIPA cell lysis buffer (Thermo Scientific, USA) containing protease and phosphatase inhibitors and concentration was determined using the Coomassie protein assay reagent (Sigma-Aldrich, USA). Equivalent amount of protein samples (40–60 μg) were subjected to SDS–PAGE, transferred onto PVDF membranes, and then incubated with primary antibodies: Nrf2 (1:250, cell signaling), GCLC, HO-1 (1:1000, Abcam, USA), NQO1 (1:1000, Santa cruz biotech, USA) overnight at 4 °C. Next day, blots were washed with TBST (tris buffered saline with tween 20) and incubated with horseradish peroxidase conjugated secondary antibody (1:3000; Sigma-Aldrich, USA) for 60 min with gentle shaking at room temperature. Blots were then washed with TBST and developed with chemiluminescence reagent (Bio-Rad, Hercules, CA) using autoradiography films (Genesee Scientific, San Diego, CA). β-actin (1:3000; Sigma-Aldrich, USA) expression was evaluated to determine equivalent loading. Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/ ) and expressed as fold change.
4
Western Blot Analysis of Protein Expression
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Western blots were carried out as previously published [25 (link), 26 (link)]. Primary antibodies were incubated overnight at 4 °C: YB-1 (ENZO Life Sciences, NY, USA) at 1:1000 dilution, P-Glycoprotein (ABCB1) rabbit monoclonal (Abcam, MA, USA) at 1:500 dilution, P-Akt (S473), β-Catenin, GSK-3β, SOX2 and GAPDH (Cell Signalling, MA, USA) at 1:1000 dilution. Secondary antibodies were horseradish peroxidase (HRP) conjugated anti-mouse and anti-rabbit antibodies (Cell Signalling, MA, USA) for use with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, MA, USA) and imaged using autoradiography films (Genesee Scientific, CA, USA). Band intensity was quantified using ImageJ software (NIH.gov ). Experiments were repeated at least 3 times.
5
Comprehensive Western Blot and qPCR Protocol
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Western blotting was performed as previously described2 (link). Primary and secondary antibodies used in this study are shown in Supplemental Table 1 . For fluorescent antibodies membranes were imaged using Odyssey infrared imaging system (Li-Cor), and for HRP conjugated antibodies, membranes were treated with ECL reagents (Bio-Rad or Millipore Sigma) developed using autoradiography film (Genesee Scientific). Band intensities were quantified using ImageJ. RNA was extracted using the RNeasy kit (Qiagen). cDNA was synthesized using the ImProm-II Reverse Transcription System (Promega). Taqman probes used for qPCR are listed in Supplemental Table 1 , and qPCR was performed using a CFX384 Touch Real-Time PCR Detection System (Bio-Rad). Actb and Gapdh were used for normalization controls. Reagent concentrations and supplier information are included in the Reagent Table .
6
Immunoblotting of YFP-tagged Proteins
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Agrobacterium tumefaciens GV2260 cultures carrying the pEG101-PvH3s-YFP constructs were infiltrated into young but fully expanded tobacco leaves at a concentration of OD600nm = 0.5. Leaf disks (1.9 cm diameter) were collected 3 days post inoculation using a cork borer, ground in liquid nitrogen and re-suspended in 100 μl 3 × Laemmli buffer containing 16% β-mercaptoethanol. The tissue was then boiled for 10 min and pelleted at a high speed for 10 min. Twenty micro liters of protein extract was applied to and separated on a 10% SDS-PAGE gel. The proteins were blotted to a PVDF membrane using a Bio-Rad Trans-Blot®TurboTM Transfer System. The membrane was blocked with 5% nonfat skim milk in 1 × Tris-saline buffer supplemented with 0.5% Tween 20 (1 × TBST). Next, the membrane was probed with anti-HA-HRP (Abcam, 1:2000) and the signal was detected with using SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA). The chemiluminescent signals were exposed to autoradiography film (Genesee Scientific, San Diego, CA) using a Kodak film processor (Kodak, A Walsh Imaging, Inc, Pompton Lakes, NJ).
7
Comprehensive Western Blot and qPCR Protocol
8
Detecting Effector Fusion Proteins
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The effector genes cloned in pEG101‐SacB/R could express the effecor‐YFP‐HA fusion proteins, which can be detected with anti‐HA antibodies by Western blot. At 20 h after Agrobacterium infiltration, the transiently expressed effecor fusion proteins were extracted by grinding three leaf discs (19 cm diameter) in 100 µL 3 × Laemmli buffer containing 16% β‐mercaptoethanol. Also, 20 µL protein samples were separated on a 10% SDS‐PAGE gel, and blotted to a PVDF membrane. The blot was probed with anti‐HA‐HRP (Sigma, St. Louis, MO; H6533; 1:500), and the signal was detected with using SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA). The chemiluminescent signals were exposed to autoradiography film (Genesee Scientific, San Diego, CA) using a Kodak film processor (Kodak, A Walsh Imaging, Inc, Pompton Lakes, NJ). Another 20 µL protein samples were also separated on a 10% SDS‐PAGE gel, and stained with comassie blue to confirm the equal loadings.
9
LPS Characterization of P. aeruginosa Strains
10
Characterizing CARMIL3 Interactome in vivo
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P0 Cas9 KI pups were intracortically injected with AAV-U6-CARMIL3Ctermsg1-HITI-myc-P2A-mCherry-SynI-Cre as described above. At P7 samples were collected and incubated with Myc beads, run out on an SDS-PAGE gel, transferred, and prepped for immunoblotting as described above. Blots were incubated overnight in Rat anti-HA (Sigma, 3F10, 1:2000), rabbit anti-Wrp (V0111)56 (link), and rabbit anti-capping protein beta (Millipore, AB6017, 1:500) at 4 °C. Membrane was then washed three times with TBST for 10 min each wash and then incubated HRP anti-Rat (GE Healthcare Life Sciences, NA935, 1:5000) and HRP anti-rabbit (GE Healthcare Life Sciences, NA934-100ΜL). Blots were developed using autoradiography film (Genesee Scientific, 30–100). Original scans of blots are included (Supplementary Figure 10 ).
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