As LC3-II is not detectable in lysates of embryonic spinal cord by Western blot, it was enriched by preventing its degradation with chloroquine, added to culture medium to a final concentration of 100 μM during the last hour of embryo culture. The spinal cords were dissected with meninges and total lysates were obtained using a buffer consisting of 68.5 mM Tris HCl at pH 6.8, 2% SDS, and protease inhibitors cOmplete™ULTRA tablets (Sigma 5892791001). Lysate was cleared using a pestle sonicator for 10 s at 30% cycle, and centrifuging at 10,000 g for 10 min at room temperature. Protein was quantified using the DCTM-protein assay kit (Bio-Rad, 5000112) using bovine serum albumin as standard. Protein was subjected to electrophoresis, transferred to a PVDF-FL membrane and analyzed using the LC3 antibody (MBL, PD014 at 1:2000 dilution overnight at 4°C) or anti-β-actin (Santa Cruz, sc-47778 at 1:10,000 for 1 h at room temperature). Secondary antibodies (IRDye® 800CW Goat anti-Rabbit (LI-COR, 92632211)and IRDye® 680RD Goat anti-Mouse IgG (LI-COR, 92568070) were diluted 1:10,000 and incubated for 1 h at room temperature. Bands were visualized in an Odyssey® CLx Imager, and quantified with the Image Studio Lite Version 5.2 software.
Complete ultra tablets
Manufactured by Merck Group
Sourced in United States
The COmplete ULTRA Tablets are a laboratory equipment product from Merck Group. They are designed for use in various laboratory applications, providing a consistent and reliable performance.
Automatically generated - may contain errors
Lab products found in correlation
Nuclei ez prep buffer, by Merck Group (3 mentions)
Phosstop, by Merck Group (2 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Casein blocker, by Bio-Rad (2 mentions)
Chemidoc mp image system, by Bio-Rad (2 mentions)
Rnasin plus, by Promega (2 mentions)
Superase in, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Dctm protein assay kit, by Bio-Rad (1 mentions)
Irdye 680rd goat anti mouse igg, by LI COR (1 mentions)
Mouse il 22 duoset elisa, by R&D Systems (1 mentions)
Ab14715, by Abcam (1 mentions)
Chemidoc touch system, by Bio-Rad (1 mentions)
Image lab software 5, by Bio-Rad (1 mentions)
Af1857, by R&D Systems (1 mentions)
Rpmi 1640 1 medium, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution 10, by Merck Group (1 mentions)
16 protocols using complete ultra tablets
1
Detecting LC3-II in Embryonic Spinal Cord
2
Quantifying Intestinal IL-22 and IL-23 Levels
3
Western Blot Analysis of Intestinal Proteins
4
Western Blot Analysis of β-Arrestin1
5
Nuclei Isolation from Xenograft Biopsies
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After a section was taken for spatial transcriptomics, the remaining xenograft biopsy tissue was thawed from OCT and washed with PBS. Nuclei were subsequently isolated using Nuclei Lysis Buffer containing Nuclei Isolation Kit: Nuclei EZ Prep Buffer (Sigma) supplemented with cOmplete ULTRA Tablets (Sigma) and SUPERase IN (Thermo) and Promega RNAsin Plus nuclease inhibitors. Tissue was minced into <1 mm pieces in 2 mL of Nuclei Lysis Buffer. Samples were transferred to a Dounce homogenizer (Kimble) and homogenized. An additional 2 mL of Nuclei Lysis Buffer was added to the sample and incubated for 5 minutes on ice. Samples were passed through a 40 μm filter into a 50 mL conical tube. Samples were centrifuged at 500 × g for 5 minutes at 4°C. The supernatant was removed and the pellet was washed with 4 mL of Nuclei Lysis Buffer containing 1% Bovine Serum Albumin for 5 minutes on ice. Samples were centrifuged at 500 × g for 5 minutes at 4°C. Samples were passed through a 5 μm filter into a 50 mL conical tube and then centrifuged again. Nuclei were resuspended in a solution containing phosphate-buffered saline (PBS), 1% BSA, and 0.1% RNAse inhibitor.
6
Cell Culture Reagents and Chemicals
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Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI 1640 (1×) Medium, L-Glutamine, penicillin/streptomycin (pen/strep), Fetal Bovine Serum (FBS) and phosphate-buffered saline (PBS) were from Gibco™ (Life Technologies, Monza MB, Italy). Trypsin-EDTA solution 10×, protease inhibitors (cOmplete™ ULTRA Tablets, cat#5892970001), Digitonin (D141), formaldehyde and NP-40 were from MERCK/Sigma-Aldrich (Milan, Italy). Bortezomib was purchased from LC Laboratories (Woburn, MA, USA).
7
Protein Extraction from HEK293 Cells and Feces
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Proteins in the HEK293 cell sample
were extracted in 100 mM Tris-HCl (pH 8.5) containing 2% sodium dodecyl
sulfate (SDS) by sonication using Bioruptor II (CosmoBio, Tokyo, Japan)
for 10 min. Protein extraction from feces with a focus on the host
proteins was performed with a slight modification in the previously
reported procedure.18 (link) Briefly, the feces
was added in tris-buffered saline (TBS) with protease inhibitors [cOmplete
ULTRA Tablets (CAT# 5892970001), Sigma-Aldrich, MO, USA], and soluble
proteins were extracted by pipetting with a tip with the tip cut off
and inverting after incubating for 30 min on ice. After centrifugation
at 15,000g for 15 min at 4 °C to remove the
pellet (bacteria and food debris), the supernatant was transferred
to a fresh tube and subjected to trichloroacetic acid precipitation
(12.5% v/v, final concentration of trichloroacetic acid), followed
by two acetone washings. The sample was redissolved in 100 mM Tris-HCl
(pH 8.5) containing 2% SDS by sonication using Bioruptor II (CosmoBio).
Protein concentration in the protein extract was determined using
a BCA protein assay kit (CAT# 23225, Thermo Fisher Scientific) and
adjusted to 1 μg/μL with 100 mM Tris-HCl (pH 8.5) containing
2% SDS.
were extracted in 100 mM Tris-HCl (pH 8.5) containing 2% sodium dodecyl
sulfate (SDS) by sonication using Bioruptor II (CosmoBio, Tokyo, Japan)
for 10 min. Protein extraction from feces with a focus on the host
proteins was performed with a slight modification in the previously
reported procedure.18 (link) Briefly, the feces
was added in tris-buffered saline (TBS) with protease inhibitors [cOmplete
ULTRA Tablets (CAT# 5892970001), Sigma-Aldrich, MO, USA], and soluble
proteins were extracted by pipetting with a tip with the tip cut off
and inverting after incubating for 30 min on ice. After centrifugation
at 15,000g for 15 min at 4 °C to remove the
pellet (bacteria and food debris), the supernatant was transferred
to a fresh tube and subjected to trichloroacetic acid precipitation
(12.5% v/v, final concentration of trichloroacetic acid), followed
by two acetone washings. The sample was redissolved in 100 mM Tris-HCl
(pH 8.5) containing 2% SDS by sonication using Bioruptor II (CosmoBio).
Protein concentration in the protein extract was determined using
a BCA protein assay kit (CAT# 23225, Thermo Fisher Scientific) and
adjusted to 1 μg/μL with 100 mM Tris-HCl (pH 8.5) containing
2% SDS.
8
Western Blot Analysis of β-Arrestin1
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One well of a 6 well plate treated with β-arrestin1 specific siRNA or control siRNA was washed with ice-cold PBS 1X, Cells were incubated for 5 mins on ice in 350 μl of pre-chilled RIPA buffer supplemented with a protease inhibitor cocktail (Complete ULTRA tablets, Mini by Sigma-Aldrich/Roche). Cells were scraped and transferred to a microcentrifuge tube. Cells were rocked at 4°C for 30 mins then centrifuged for 15 mins at 14,000 rpm at 4°C. Supernatants were mixed with sample buffer, boiled for 5 mins at 95°C and resolved by SDS-PAGE (4%–15% Tris-HCl polyacrylamide gel). Proteins were transferred to a nitrocellulose membrane by Semidry transfer, blocked with 1X TBS 1% casein blocker (BioRad #161–0782) for 1hr at RT then incubated with anti-β-arrestin1/ 2 (1:500) O/N at 4°C. The membrane was washed 4 times with 1X TBS-T then incubated with goat anti-rabbit Alexa 680 (1:5,000) at RT for 1 hr, washed thoroughly with 1X TBS-T and imaged using the ChemiDoc MP Image system (BioRad). The membrane was then probed with anti-GAPDH (1:10,000) O/N at 4°C, washed 4 times with 1X TBS-T then incubated with goat anti-rabbit IRDye CW 800 (1:5,000) for 1 h at RT, washed thoroughly with 1X TBS-T and imaged using the ChemiDoc MP Image system (BioRad).
9
Kidney Nuclei Isolation Protocol
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Nuclei were isolated using Nuclei Lysis Buffer containing Nuclei Isolation Kit: Nuclei EZ Prep Buffer (MilliporeSigma) supplemented with cOmplete ULTRA Tablets (MilliporeSigma) and SUPERase IN (Thermo Fisher Scientific) and Promega RNAsin Plus nuclease inhibitors. Kidneys were minced into less than 1 mm pieces in 2 mL Nuclei Lysis Buffer. Samples were transferred to a dounce homogenizer (Kimble) and homogenized. An additional 2 mL Nuclei Lysis Buffer was added to the sample and incubated for 5 minutes on ice. Samples were passed through a 40 μm filter into a 50 mL conical tube. Samples were centrifuged at 500g for 5 minutes at 4°C. The supernatant was removed, and the pellet was washed with 4 mL Nuclei Lysis Buffer containing 1% bovine serum albumin for 5 minutes on ice. Samples were centrifuged at 500g for 5 minutes at 4°C. Samples were passed through a 5 μm filter into a 50 mL conical tube and then centrifuged again. Nuclei were resuspended in a solution containing PBS, 1% BSA, and 0.1% RNAse inhibitor.
10
Exosomal Biomarker Quantification Protocol
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After having been thawed, samples received M-PER mammalian protein extraction reagent to lyse exosomes (ThermoFisherScientific), containing three times the suggested concentrations of protease and phosphatase inhibitors (complete™ ULTRA Tablets; MilliporeSigma, Burlington, MA). These mixtures were used to measure biomarker concentrations, using a site-specific Simoa HD-1 analyzer (Quanterix, Lexington, MA), an ultrasensitive paramagnetic bead-based enzyme-linked immunosorbent assay, according to the manufacturer's protocol. Coefficient of variation values were no higher than 30% for all analytes. Samples were randomized over plates, with laboratory scientists blinded to participant groups. For each sample, total and neuronal-enriched exosomal samples were run in the sample plate. All assays were run in duplicate.
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