Western Blotting was performed as previously described [73 (link)]. Briefly, protein separation from retinas (N = 5) were performed on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). This was followed by protein electrotransfer to polyvinylidene difluoride (PVDF) membrane (IPFL10100; Merck Millipore, Milan, Italy). Blocking of membranes was performed with a 5% non-fat dry milk/Tris Buffered Saline (TBS) solution for 1 h. Then, membranes were incubated overnight at 4 °C with the following primary antibodies, all diluted in 3% blocking solution: anti-Iba1 (3 µg/mL; ab5076; Abcam; PLC., Cambridge, UK); anti-LC3B (1 µg/mL; ab48394; Abcam; PLC., Cambridge, UK), and β-actin (1:1000; sc47778; Santa Cruz Biotechnology, Dallas, TX, USA). Blots were then incubated with horseradish peroxidase-conjugated secondary anti-rabbit, anti-goat, and anti-mouse antibodies (all 1:10000; respectively, sc-2004, sc-2020, and sc-2005; Santa Cruz Biotechnology, Dallas, TX, USA), for 1 h at room temperature. Iba1 and LC3B immunoreactive bands were detected by using an enhanced chemiluminescence system (35055; Thermo Fisher, Waltham, MA, USA). Then, these were quantized with VisionWorks Life Science Image Acquisition and Analysis software (UVP, Upland, CA, USA), normalized with β-actin protein levels and expressed as densitometric units (DU).
Ipfl10100
Manufactured by Merck Group
Sourced in Italy
The IPFL10100 is a laboratory equipment product manufactured by the Merck Group. It serves as a device for performing various laboratory functions, but a detailed description without interpretation or extrapolation cannot be provided.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico chemiluminescent substrate, by Bio-Rad (3 mentions)
Anti actin, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Sars cov 2 nucleoprotein, by Sino Biological (2 mentions)
Anti actin, by Transgene (2 mentions)
Ab5076, by Abcam (1 mentions)
Ab48394, by Abcam (1 mentions)
β actin, by Analytik Jena (1 mentions)
Anti mouse sc 2005, by Santa Cruz Biotechnology (1 mentions)
Ab55391, by Abcam (1 mentions)
Np0323, by Thermo Fisher Scientific (1 mentions)
Ab37073, by Abcam (1 mentions)
Pa1 16927, by Thermo Fisher Scientific (1 mentions)
Image quant las400 mini, by GE Healthcare (1 mentions)
Anti rabbit igg hrp antibody, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Sars cov 2 spike, by Sino Biological (1 mentions)
Gapdh, by Transgene (1 mentions)
9 protocols using ipfl10100
1
Western Blot Analysis of Retinal Proteins
2
Western Blotting Analysis of Protein Targets
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Western Blotting assay was performed in a 12% PAGE separation gel, electro-transferring 30 μg of protein sample onto a PVDF membrane (IPFL10100 Merck Millipore, Italy), blocked for 1 h at room temperature with 5% non-fat dry milk (EMR180500 Euroclone, Italy). Then blots were incubated over-night with the following specific primary antibodies: anti-MC5R (sc-28994 Santa Cruz, United States), anti-GPR14 for Urotensin II receptor detection (sc-288998 Santa Cruz, United States), anti KIR 6.1 (P0874 Sigma, Italy), and anti-actin (sc-8432 Santa Cruz, United States). This step was followed by incubation for 1 h at room temperature with horseradish peroxidase-conjugated secondary anti-rabbit (sc-2004 Santa Cruz, United States) or anti-mouse (sc-2005, Santa Cruz, United States) antibodies. The signal was expressed as Densitometric Units (D.U.).
3
Western Blot Analysis of KRas Protein
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Cells were cultured in 6-well plates for 24–48 hr before lysing with a RIPA buffer (Thermo Scientific, 89901) supplemented with an inhibitor cocktail (ThermoFisher, 88668). Cell lysates were then harvested, sonicated, and centrifuged. The supernatant is assayed using BCA and analyzed using a Bris-Tris gel (4–12%, ThermoFisher NP0323). Protein transfer was performed on a low fluorescence PVDF membrane (EMD Millipore, IPFL10100). The membrane was then immunostained for fluorescence detection using a Li-COR Odyssey. The antibodies used for this study were: KRas (mouse monoclonal, Abcam ab55391, RRID:AB_941040 , used at 1:200 dilution), Tubulin (mouse monoclonal, ThermoFisher 32–2600, RRID:AB_86547 , used at 1:500 dilution).
4
Protein Expression Analysis by Western Blot
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Proteins were separated on a 12% PAGE separation gel and then electrotransferred onto a PVDF membrane (IPFL10100 Merck Millipore, Italy). The membrane was blocked for 1 hour at room-temperature with 5% nonfat dry milk and then incubated overnight with the following primary specific antibodies: anti-actin (sc-8432 Santa Cruz, USA), anti-HCA2 (A02511 BosterBio, USA), anti-eukaryotic translation initiation factor 2-α kinase (PERK) (sc-377400Santa Cruz, USA), anti-phospho-PERK (pPERK) (sc-32557 Santa Cruz, USA), anti-inositol requiring enzyme 1 (IRE1) (ab37073abcam, Italy) and anti-phospho-IRE1 (pIRE1) (PA1-16927 ThermoFisher, Italy). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5000 ADI-SAB-300) and anti-mouse (1:5000 ADISAB-110 Enzo, Italy) were used as secondary antibodies and incubated for 1 hour at room temperature. The chemiluminescent signals were revealed and expressed as Densitometric Units (D.U.).
5
SARS-CoV-2 Nucleoprotein Detection
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The cell lysates were collected and run on the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, IPFL10100). After blocking with bovine serum albumin, primary antibody incubation and secondary antibody incubation, the desired bands were detected using a Super Signal West Pico Chemiluminescent substrate (Bio‐Rad). The following antibodies were used: SARS‐CoV‐2 nucleoprotein (Sino Biological, 40588‐T62), actin (TransGen Biotech, HC201‐02), anti‐mouse IgG (TransGen Biotech, HS201‐01), and anti‐rabbit IgG (TransGen Biotech, HS101‐01).
6
Western Blot Analysis of Melanocortin Receptors
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ARPE-19 cells were collected in RIPA Buffer (Sigma-Aldrich R0278) and protease inhibitor cocktail (Roche 11873580001). Equal amounts of protein (20 µg) were loaded and analyzed by SDS-PAGE on 4–12% SDS-Polyacrylamide gel electrophoresis, and the proteins electroblotted onto polyvinylidene difluoride membranes (PVDF; Millipore IPFL10100) by wet transfer. Membranes were incubated overnight at 4°C with antibodies against MCR1 (Abcam ab180776), MCR2 (Abcam ab180793), MCR3 (Abcam ab203671), MCR4 (Abcam ab24233), MCR5 (Abcam ab133656), VEGF (Santa Cruz Biotechnology sc-57496), NF-kB (Abcam ab32536), Cyp2E1 (Abcam, ab28146) and β-actin (Santa Cruz Biotechnology sc-8432), which was the loading control. Finally, the membranes were incubated for 2 hours at room temperature with anti-mouse (Santa Cruz Biotechnology sc-2005) or anti-rabbit IgG-HRP antibodies (Santa Cruz Biotechnology sc-2004). The bands were visualized with ECL (Pierce, Thermo Scientific, 32132) and detected with Image Quant LAS-400 mini (GE Healthcare, Uppsala, Sweden). Protein levels were quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA).
7
SARS-CoV-2 Protein Expression Analysis
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Cells were harvested and boiled in 2X lammili sample buffer containing 10% βME (Sigma, M3148). Cell lysates were separated by 9% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, IPFL10100). After being blocked with 5% BSA in TBS buffer containing 0.05% Tween 20, the blot was probed with indicated first antibodies and the horseradish peroxidase (HRP)-conjugated secondary antibody sequentially. Protein bands were detected by SuperSignal West Pico Chemiluminescent substrate (Bio-Rad). The following antibodies were used: ACE2 (Abcam, ab108209), SARS-CoV-2 Nucleoprotein (Sino Biological, 40,588-T62), SARS-CoV-2 Spike (Sino Biological, 40,591-MM42), Actin (TransGen Biotech, HC201), GAPDH (TransGen Biotech, HC301), AT-1R (Abcam, ab18801), AT-2R (Abcam, ab92445), anti-mouse IgG (TransGen Biotech, HS201–01) and anti-rabbit IgG (TransGen Biotech, HS101–01).
8
Immunoblotting for SARS-CoV-2 Proteins
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Cell lysates were boiled in 2× Laemmli sample buffer containing 10% βME (Sigma, M3148). Cell lysates were analyzed by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, IPFL10100). After blocking with 5% BSA in TBS buffer containing 0.05% Tween 20, the blot was sequentially probed with primary antibodies and the horseradish peroxidase (HRP)-conjugated secondary antibody. Protein bands were detected by SuperSignal West Pico Chemiluminescent substrate (Bio-Rad). The following antibodies were used: anti-ACE2 (Abcam, ab108209), anti-SARS-CoV-2 nucleoprotein (Sino Biological, 40588-T62), anti-p-hRIPK1 (Cell Signaling Technology, 65746), anti-RIPK1 (Cell Signaling Technology, 3493), anti-EGFR (Cell Signaling Technology, 4267), anti-NSP12(Cell Signaling Technology), anti-Flag (Sigma, F1804), anti-Myc (Sigma, C3956), anti-Strep tag II (Abcam, ab76949), anti-Actin (TransGen Biotech, HC201) and anti-Tubulin (TransGen Biotech, HC101).
9
Immunoprecipitation and Western Blot Analysis
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In immunoprecipitation, HEK-293T cells were harvested with 1% NP-40 lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 5 mM glycerophosphate, 5 mM NaF) at 4 °C for 30–60 min. The lysates were centrifuged at 12,000 rpm for 10 min at 4 °C, keeping the supernatant and incubating with the indicated antibodies and Protein G Agarose (Thermo, 20397). After the incubation, beads were washed three times with lysis buffer, and then eluted with 2× Laemmli sample buffer.
Boiled cell lysates were separated by SDS–PAGE gels. Then transferred it onto polyvinylidene difluoride (NC) membranes (Millipore, IPFL10100). Blocking with 5% BSA in TBST buffer, then the blot was orderly probed with primary antibodies and the horseradish peroxidase (HRP)-conjugated secondary antibody. Protein bands were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, 34580). The following antibodies were used: anti-His (TransGen Biotech, F1804), anti-FLAG (TransGen Biotech, F1804), anti-Strep tag II (Abbkine, 8C12), anti-GFP (TransGen Biotech, HC101), anti-Mouse IgG (TransGen Biotech, HS201-01), anti-Rabbit IgG (TransGen Biotech, HS101-01) and anti-Actin (TransGen Biotech, HC201).
Boiled cell lysates were separated by SDS–PAGE gels. Then transferred it onto polyvinylidene difluoride (NC) membranes (Millipore, IPFL10100). Blocking with 5% BSA in TBST buffer, then the blot was orderly probed with primary antibodies and the horseradish peroxidase (HRP)-conjugated secondary antibody. Protein bands were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, 34580). The following antibodies were used: anti-His (TransGen Biotech, F1804), anti-FLAG (TransGen Biotech, F1804), anti-Strep tag II (Abbkine, 8C12), anti-GFP (TransGen Biotech, HC101), anti-Mouse IgG (TransGen Biotech, HS201-01), anti-Rabbit IgG (TransGen Biotech, HS101-01) and anti-Actin (TransGen Biotech, HC201).
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