The following antibodies were used in the study: CDK4 (Abcam, Cambridge, United Kingdom, 1:1,000), CDK6 (Abcam, Cambridge, United Kingdom, 1:50,000), Rb (Abcam, Cambridge, United Kingdom, 1:2000), phospho-Rb S780 (Abcam, Cambridge, United Kingdom, 1:1,000), LC3B (Cell Signaling Technology, Danvers, MA, United States, 1:1,000), P62 (Cell Signaling Technology, Danvers, MA, United States, 1:1,000), LAMP2 (Cell Signaling Technology, Danvers, MA, United States, 1:1,000), β-actin (Beyotime, Shanghai, China, 1:1,000). Abemaciclib and Abemaciclib metabolite M2 were purchased from MedChemExpress (Monmouth Junction, NJ, United States, LY-2835219, LSN2839567). Compounds LPM3770277, LPM3770278, and LPM3770304 were synthesized by Shandong Luye Pharmaceutical Corporation (Yantai, China).
Lamp2
Manufactured by Cell Signaling Technology
Sourced in United States, China, Denmark
LAMP2 is a lysosome-associated membrane glycoprotein that plays a role in the fusion of lysosomes with autophagosomes during the process of autophagy. It is commonly used as a marker for autophagic structures.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (4 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Ampkα, by Cell Signaling Technology (3 mentions)
Alexa 594 red conjugated anti rabbit igg, by Vector Laboratories (2 mentions)
Fluorescein isothiocyanate green labeled anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Tcs sp5 confocal microscope, by Leica (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Foxo4, by Cell Signaling Technology (2 mentions)
Procaspase 3, by Cell Signaling Technology (2 mentions)
Foxo1, by Santa Cruz Biotechnology (2 mentions)
α actin, by Agilent Technologies (2 mentions)
Pico tag reverse phase column, by Waters Corporation (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
18 protocols using lamp2
1
Antibodies for Cell Cycle and Autophagy Analysis
2
Quantifying Autophagy in MC3T3-E1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MC3T3-E1 cells cultured on culture slides (BD Falcon Labware, REF 354108) were fixed in methanol at −20 °C for 3 min. Cells were washed with PBS, blocked with 10% bovine serum albumin (Sigma-Aldrich) in PBS for 10 min, and incubated with primary antibody, Lamp2 (Cell Signaling Technology) and LC3 (Cell Signaling Technology), in blocking buffer at 4 °C overnight. Cells were hybridized with secondary antibodies, Alexa 594 (red)-conjugated anti-rabbit IgG (Vector Laboratories Inc., Burlingame, CA, USA) and fluorescein isothiocyanate (green)-labeled anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), for 1 h at room temperature. The coverslips on glass slides were mounted using Vectashield mounting medium (Vector Laboratories Inc.). Cells were observed under a Leica TCS SP5 confocal microscope (Leica, Microsystems CMS GmbH, Wetzlar, Germany). Cells were stained with 4,6-diamidino-2-phenylindole (DAPI, Santa Cruz Biotechnology) for 10 min. Immunofluorescence quantities of both LC3 and Lamp2 indicate the level of autophagosomes or autophagy.
3
Protein Expression Analysis in Muscle
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen longissimus dorsi muscle samples were homogenized and centrifuged at 10,000 g for 10 min at 4°C. The protein concentration was determined in the supernatant by the Bradford method
[31 (link)]. Equal amounts (50 μg) of extracted protein were electrophoretically separated in polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA), which were incubated with appropriate primary antibodies followed by appropriate secondary antibodies as previously described
[33 (link)]. Blots were developed using an enhanced chemiluminescence kit (Amersham), visualized, and analyzed using a ChemiDoc-It Imaging System (UVP, Upland, CA). The protein abundance of each signaling components was normalized with β-actin abundance in the samples. Primary antibodies that were used in the immunoblotting were MuRF1, atrogin-1, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), rpS6, eIF4E, Lamp-2, ULK1, and LC3 (Cell Signaling Technology, Danvers, MA).
[31 (link)]. Equal amounts (50 μg) of extracted protein were electrophoretically separated in polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA), which were incubated with appropriate primary antibodies followed by appropriate secondary antibodies as previously described
[33 (link)]. Blots were developed using an enhanced chemiluminescence kit (Amersham), visualized, and analyzed using a ChemiDoc-It Imaging System (UVP, Upland, CA). The protein abundance of each signaling components was normalized with β-actin abundance in the samples. Primary antibodies that were used in the immunoblotting were MuRF1, atrogin-1, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), rpS6, eIF4E, Lamp-2, ULK1, and LC3 (Cell Signaling Technology, Danvers, MA).
4
Western Blot Analysis of Hepatic Stellate Cell Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LX2 cells were treated according to the experimental design. LX2 cell lysates were collected in ice-cold RIPA buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Proteins (5 µg) were separated via SDS-PAGE using 10% gels, and electrotransferred to PVDF membranes (MilliporeSigma), which were incubated with anti-collagen-I (COL-I; cat. no. AF7001; 1:1,000; Affinity Biosciences), α-smooth muscle actin (α-SMA; cat. no. 19245; 1:1,000; Cell Signaling Technology, Inc.), LC3 (cat. no. 2775; 1:1,000; Cell Signaling Technology, Inc.), P62 (cat. no. 8025; 1:1,000; Cell Signaling Technology, Inc.), LAMP2 (cat. no. 49067; 1:1,000; Cell Signaling Technology, Inc.) and β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc.) primary antibodies at 4°C overnight. Subsequently, membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-Rabbit IgG secondary antibody (cat. no. SA00001-2; 1:2,000; ProteinTech Group, Inc.) for another 2 h at room temperature. All blots were visualized using enhanced chemiluminescence (ECL) reagents (Thermo Fisher Scientific, Inc.), and the scanned band images were semi-quantified using ImageJ software version 1.8 (National Institutes of Health).
5
Investigating Cellular Mechanisms in Disease
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRIzol was purchased from Sigma (St Louis, Missouri, USA). TMR red-labeled In-Situ Cell Death Detection reagent and sTREM-1, the sSirt6 enzyme-linked immune sorbent assay (ELISA) kit were purchased from Roche Applied Science (Indianapolis, USA). Monoclonal antibodies against CD62-PE, CD31-PE, CD42-FITC, and IgG-PE were purchased from BD (Shanghai, China), and monoclonal rabbit antibodies against Sirt6, TREM-1, LC3, BECN1, LAMP2, p62, Caspase-1, and GAPDH were purchased from Cell Signaling (Denver, Colorado, USA). Fetal bovine serum (FBS) and Lipofectamine® 2000 Transfection Reagent were purchased from Invitrogen (Carlsbad, CA, USA). The cDNA Synthesis Kit and Premix Ex Taq SYBR Green PCR Kit were purchased from Takara (Shiga, Japan). The adv-Sirt6, LV-Sirt6 shRNA, adv-TREM-1, LV-TREM-1 shRNA, LV-LC3 shRNA, and GFR, GRP double labeled adv-LC3 were purchased from HanBio. (Shanghai, China). Other unmentioned reagents were purchased from Shenggong Bio. (Shanghai, China).
6
Measuring Muscle Amino Acid Metabolism
7
Measuring Muscle Amino Acid Metabolism
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immediately following euthanasia, LD samples were stored at −70°F until processing. Free intracellular AA concentrations in muscle were determined by high-performance liquid chromatography (HPLC; PICO-TAG reverse-phase column; Waters, Mildford, MA,) using an analytical method based on deproteinization and derivatization of AA with phenylisothiocyanate, as previously described (18 (link)).
Amino acid transporter and intracellular markers of protein degradation and autophagy were measured by immunoblotting analysis, as previously described (8 (link), 31 (link)). The antibodies used in the immunoblotting process were PKB (total and Ser473, Cell Signaling Technology, Beverly, MA), AMPK-α (total and Thr172, Cell Signaling Technology, Beverly, MA), FOXO1 (total protein, Santa Cruz Technology, Santa Cruz, CA; Ser256, Cell Signaling Technology), FOXO4 (total protein and Ser262, Cell Signaling Technology, Beverly, MA), pro-caspase 3 (total protein, Cell Signaling Technology, Beverly, MA), MuRF1 (ECM Biosciences, Versailles, KY), atrogin-1 (ECM Biosciences, Versailles, KY), LAT1 (Cell Signaling Technology, Beverly, MA), SNAT2 (Aviva System Biology, San Diego, CA.), LC3 (Cell Signaling Technology, Beverly, MA), β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), α-Actin (40 KDa; Dako-Cytomation, Glostrup, Denmark) and LAMP-2 (Cell signaling Technology, Danvers, MA)
Amino acid transporter and intracellular markers of protein degradation and autophagy were measured by immunoblotting analysis, as previously described (8 (link), 31 (link)). The antibodies used in the immunoblotting process were PKB (total and Ser473, Cell Signaling Technology, Beverly, MA), AMPK-α (total and Thr172, Cell Signaling Technology, Beverly, MA), FOXO1 (total protein, Santa Cruz Technology, Santa Cruz, CA; Ser256, Cell Signaling Technology), FOXO4 (total protein and Ser262, Cell Signaling Technology, Beverly, MA), pro-caspase 3 (total protein, Cell Signaling Technology, Beverly, MA), MuRF1 (ECM Biosciences, Versailles, KY), atrogin-1 (ECM Biosciences, Versailles, KY), LAT1 (Cell Signaling Technology, Beverly, MA), SNAT2 (Aviva System Biology, San Diego, CA.), LC3 (Cell Signaling Technology, Beverly, MA), β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), α-Actin (40 KDa; Dako-Cytomation, Glostrup, Denmark) and LAMP-2 (Cell signaling Technology, Danvers, MA)
8
Analyzing Autophagy in MDA-MB-231 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, the MDA-MB-231 cell model expressing mRFP-LC3, mRFP-LC3, were cultured on cover slides in 10% FBS-supplemented DMEM. After synchronization, cells were cultured in complete medium for 12 h, followed by 0.1% FBS medium for another 12 h. The cells were fixed with 4% paraformaldehyde (Sigma Aldrich Corporation, 158127) in PBS (Gibco, 8117296) at room temperature (RT) for 15 min, followed by permeabilization with 0.25% Triton X-100 (Beyotime, ST795) at RT for 15 min and staining with LAMP2 (1:100; Cell Signaling Technology, D3U4 C) for 12 h at 4°C. The slides were washed three times with PBS and stained with corresponding secondary IgG for 60 min at RT. The slides were washed three times with PBS and once with ddH2O, and then fixed in glycerin. Cell images were captured using an inverted Nikon fluorescence microscope (A1 R). For the quantification of autophagic cells, puncta were determined in 20 random cells per slide.
9
GH3 Cell Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GH3 cells grown on culture slides (BD Falcon Labware, REF 354108) were permeabilized and fixed in methanol at −20°C for 3 min. Cells were washed with phosphate-buffered saline (PBS), blocked with 10% bovine serum albumin (Sigma) with PBS for 10 min, and incubated with primary antibody in blocking buffer for 1 h at room temperature (RT). Cells were hybridized with secondary antibodies for 1 h at RT. The coverslips were mounted on glass slides using Vectashield mounting medium (Vector Labs Inc., Burlingame, CA, USA). Cells were viewed under a Leica TCS SP5 confocal microscope (Leica, Microsystems CMS GmbH, Germany). The following primary antibodies were used: Lamp2 (Cell Signaling Technology) and LC3 (Cell Signaling Technology). The following secondary antibodies were used: Alexa 594 (red)-conjugated anti-rabbit IgG (Vector Laboratories Inc.) and fluorescein isothiocyanate (green)-labeled anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).
10
Western Blot Analysis of Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were incubated with cell lysis buffer (Cell Signaling, Danvers, USA) containing inhibitor cocktail of protease and phosphatase (Sigma-Aldrich) on ice for 30 min. The protein sample (25-40 μg) was run on 6%, 10%, or 15% Tris-glycine gels, transferred to polyvinylidene fluoride (PVDF) membrane, blocked in 5% nonfat dry milk at room temperature (RT) for 2 h, and incubated with the primary antibody at 4°C overnight and secondary antibody at RT for 1 h. Membranes were developed with electrochemiluminescence (Tanon, Shanghai, China) and subsequently autoradiographied (Tanon). Quantification of the results adjusted to internal reference protein (β-actin) was performed by ImageJ (Standard Edition, Bethesda, USA). Primary antibodies for phospho-mTOR (#s6448, 1 : 1000), mTOR (#2983P, 1 : 1000), phospho-P70S6K (#9234, 1 : 1000), RAPTOR (#2280, 1 : 1500), P70S6K (#2708, 1 : 1000), and LAMP2 (#49067, 1 : 1000) were from Cell Signaling Technology. Primary antibodies for LC3 (#12135-1-AP, 1 : 1000), p62/SQSTM1 (#18420-1-AP, 1 : 10000), LAMP1 (#21997-1-AP, 1 : 1000), and BECN1 (#11306-1-AP, 1 : 1000) were from Proteintech Group (Wuhan, China). Primary antibodies for NRF2 (#SC13032, 1 : 500) and β-actin (#SC1616, 1 : 5000) were from Santa Cruz Biotechnology (Santa Cruz, USA). Secondary antibodies were from Thermo Fisher Scientific.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!