Cells were harvested in sample buffer (0.1 M Tris [pH 6.8], 4% SDS, 4 mM EDTA, 286 mM 2-mercaptoethanol, 3.2 M glycerol, 0.05% bromophenol blue), and proteins were analyzed as previously described (8 (link)). Commercial antibodies used in this study are as follows: MNV Propol (9 (link)) ATG16L1 and LC3B (Sigma-Aldrich); GBP1-5 and Actin-HRP (Santa Cruz Biotechnology); HA (the Frank W. Fitch Monoclonal Antibody Facility, The University of Chicago); Flag-M2 (Thermo Fischer); WIPI2 (Abcam); HRP Goat anti-mouse and HRP Donkey anti-rabbit (Bio Legend); and HRP Donkey anti-goat (Jackson Immuno Research).
Wipi2
Manufactured by Abcam
Sourced in Germany, United States
WIPI2 is a protein that is involved in the regulation of autophagy, a cellular process that degrades and recycles damaged or unwanted cellular components. WIPI2 plays a role in the formation of autophagosomes, which are the double-membrane vesicles that engulf the cargo to be degraded.
Automatically generated - may contain errors
Lab products found in correlation
Atg16l1, by Abcam (2 mentions)
Donkey anti goat hrp, by Jackson ImmunoResearch (1 mentions)
Gapdh, by ZenBio (1 mentions)
Ubiquitin, by Abcam (1 mentions)
β actin, by ZenBio (1 mentions)
Phospho sqstm1 thr269 ser272 specific antibody, by PhosphoSolutions (1 mentions)
Flag tag (flag), by ProSpec (1 mentions)
Myc tag, by BioLegend (1 mentions)
Beclin 1, by Proteintech (1 mentions)
Water soluble cholesterol, by Merck Group (1 mentions)
Anti actb β actin, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Anti rab11, by Cell Signaling Technology (1 mentions)
Atg16l1, by Cell Signaling Technology (1 mentions)
Anti atg9a, by Abcam (1 mentions)
Bafa1, by Santa Cruz Biotechnology (1 mentions)
Flag tag m2, by Merck Group (1 mentions)
Atg12, by Cell Signaling Technology (1 mentions)
Anti pi 3 p, by Echelon Biosciences (1 mentions)
9 protocols using wipi2
1
SDS-PAGE Protein Analysis Protocol
2
Protein Fractionation and Immunoblotting
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The total protein extract was prepared by homogenizing the cells in a 1× SDS sample buffer. The NP-40-soluble and -insoluble protein fractions from the cells were prepared as previously described [34 (link)]. Immunoprecipitation and western blot were carried out as previously described [34 (link)]. Primary antibodies against the following proteins were used for western blot analysis: K48-linked Ub chain-specific antibody (Millipore, 05-1307); K63-linked Ub chain-specific antibody (Abcam, ab179434); ubiquitin (Abcam, ab134953); Phospho-SQSTM1 (Thr269/Ser272)-specific antibody (Phosphosolutions, P196-269); SQSTM1 (Santa Cruz Biotechnology (sc-28359); ATG5 (Cell Signaling Technology, 12994); LC3B (Cell Signaling Technology, 3868); ATG16L1 (Abcam, ab187671); WIPI2 (Abcam, ab105459); Beclin1 (Proteintech, 11306-1-AP); GFP (Rockland, 600-101-215); FLAG tag (Prospec, ANT-146-b); Myc Tag (Biolegend, MMS-150R); GAPDH (Zen Bioscience, 200306); β-actin (Zen Bioscience, 200068-6D7). See Supplementary Table S2 for further details and dilutions of all antibodies.
3
Analyzing Membrane Trafficking Pathways
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The chemicals used in this study were: MBCD (Sigma, C4555), cholesterol-water soluble (Sigma, C4951), Baf A1 (Santa Cruz, CAS 88899-55-2), Wort (Santa Cruz, CAS 19545-26-7), cholera toxin subunit B conjugated with Alexa Fluor 594 (CTxB, Invitrogen, C34777), DAB (Dako, K401011), NEM (Sigma, E3876), DTT (Sigma, D9779), human transferrin peroxidase (Rockland antibodies and assays, 009-0334). The antibodies used were: anti-MAP1LC3B/LC3B (Sigma, L7543), anti-ACTB/β-actin (Sigma, A5441), anti-TFRC/TFRC (Invitrogen, 136,800), anti-VAMP3 (Santa Cruz, sc-514843), anti-CAV1/caveolin-1 (BD Pharmingen, 610,060), anti-ATG9A (Abcam, ab108338), anti-RAB11 (Cell Signaling Technology, 5589), mouse anti-STX6 (Invitrogen, 701,823), WIPI2 (Abcam, ab105459), and ATG16L1 (Cell Signaling Technology, 8089).
4
Quantification of Autophagy-related Organelles
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Cells were fixed with 4% Pfa, permeabilized with 0.2% Triton, blocked in 1% BSA and were stained with anti-PI(3)P (Echelon), LBPA (Echelon), Atg12 (Cell Signaling #2011), WIPI2 (Abcam), Flag-tag M2 (Sigma), and Myc-tag 9B11 (Cell Signaling) and imaged on upright and inverted confocal microscopes made by Zeiss (Göttingen, Germany) including Zeiss 510 and Zeiss 780. 40× and 63× oil objectives were used. For quantification, at least 30 transfected cells imaged from at least three separate experiments were examined and categorized as indicated (i.e. no rings vs. rings).
5
Autophagy and Lipophagy Assays in Hepa1-6 Cells
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Hepa1-6 cells were treated with FA or FA plus CK (35 μM) for 24 h. The cells were fixed and permeabilized. For autophagy analysis, the cells were treated with WIPI2 (Abcam, 1:100) antibodies or FIP200 (CST, 1:100) antibodies for 1 h, and then treated with Alexa Fluor 568-conjugated AffiniPure goat anti-rabbit IgG (Invitrogen, A11011, 1:100) at room temperature for 1 h. For lipophagy analysis, the cells were incubated with LC3 (CST, 1:100) antibodies or LAMP1 (CST, 1:100) antibodies for 1 h. After three washes, the cells were treated with Alexa Fluor 568-conjugated AffiniPure goat anti-rabbit IgG and BODIPY 493/563 (Thermo, D3922, 1:1000) at room temperature for 1 h. For staining the GRs, the cells were treated with dexamethasone (100 nM, D1756, Sigma-Aldrich) or CK for 2 h. Further details about the methods are included in the Supporting Information (Appendix A ).
6
Autophagy Pathway Regulation Analysis
7
Autophagy regulation via p62 phosphorylation
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ULK1(Sigma-Aldrich, #A7481), p62(Progen Biotechnik, #GP62-C), p-p62 Ser403(Millipore, #MABC186), β-Actin(Cell Signaling Technology, #3700), FLAG-M2(Sigma-Aldrich, #F1804), myc(Cell Signaling Technology, 9B11, #2276), Ubiquitin(Dako, #Z0458; Abcam, #ab7780; Biomol, clone FK2, #PW8810), LC3B(Cell signaling, #2775), WIPI2(abcam, #ab101985), Rab7(Cell signaling, #9367), LAMP2(DSHB, #H4B4), GAPDH(Chemicon, #MAB374), polyQ(Merck Millipore, #MAB1574), GFP(Life technologies, #A11122), DsRed(Clontech lab, #632496) were purchased from the indicated suppliers. Anti–phosphorylated p62 polyclonal antibody was raised in rabbits using the peptide SMGF(pS) DEGGWLTRC as an antigen by Abgent and Cocalico Biologicals.
8
Autophagy Regulation: Antibody Profiling
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The primary antibodies used were as follows: ACTB (Sigma-Aldrich, SAB5500001), ATG16L1 (Abcam, ab187671), ATG16L1 (phospho-S278; Abcam, ab192542), ATG5 (nanoTools, 0262–100), ATG7 (Abcam, ab133528), BECN1 (Cell Signaling Technology, 3495), cl-CASP3 (Cleaved-Asp175; Cell Signaling Technology, 9661), FLAG (Sigma-Aldrich, F3165), GST (Santa Cruz Biotechnology, sc-138), HA (Abcam, ab9110), His (Santa Cruz Biotechnology, clone H-3), HSPD1/HSP60 (Santa Cruz Biotechnology, sc-13115), LMNA/lamin A/C (Santa Cruz Biotechnology, sc-376248), LAMP1 (Cell Signaling Technology, 9091), LC3B (Cell Signaling Technology, 2775), MOAP1 (Sigma-Aldrich, HPA000939), MYC (Santa Cruz Biotechnology, sc-40), NBR1 (Novus Biologicals, H00004077-M01), SQSTM1/p62 (Abcam, ab56416), ABCD3/PMP70 (Abcam, ab109448), SEC22B (Abcam, 181076), STX17 (MBL International, PM076), TFR2 (Abcam, 185550), TOMM20 (Santa Cruz Biotechnology, sc-11415), TUBA/tubulin (Santa Cruz Biotechnology, sc-8035), ULK1 (Cell Signaling Technology, 8054), ULK1 (phospho-S317; Cell Signaling Technology, 12753), WIPI2 (Abcam, ab105459).
9
Colorectal Cancer Protein Expression Analysis
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First, colorectal cancer cells were treated by lysis at 4°C using RIPA lysis solution (Beyotime Biotechnology, Shanghai, China) containing fresh protease and phosphatase inhibitors, and 12,000g lysate was collected. After being freezing centrifuged for 20 minutes, the supernatant was removed to collect total cellular proteins. Antibodies used for Western blot were WIPI2 (1:1000), ACSL4 (1:5000) and GPX4 (1:1000), all purchased from Abcam, USA, and β-actin (1:1000), purchased from Beyotime Biotechnology, Shanghai, China. Thereafter, PVDF membranes were incubated with peroxidase-conjugated secondary antibody (1:1000, Beyotime Biotechnology) at 37°C for 1h. Finally, the signal was visualized using Enhanced chemiluminescence and the images were analyzed using ImageJ analysis software. All experiments were repeated at least 3 times.
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