Rabbit anti gst

Primary antibodies used in this study for immunofluorescence are the following: rabbit anti-vasa (1:5000; R. Lehmann), guinea pig anti–phospho–myosin II (1:1000; R. Ward), chicken anti-vasa (1:500; R. Lehmann), and rabbit anti-RhoGEF2 (1:2500; J. Grosshans).
Secondary antibodies used in this study for immunofluorescence are the following: Cy3 AffiniPure Donkey Anti-Rabbit IgG (immunoglobulin G) (Jackson ImmunoResearch, 711-165-152), Cy3 AffiniPure Donkey Anti-Guinea Pig IgG (Jackson ImmunoResearch, 706-165-148), Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (Jackson ImmunoResearch, 711-545-152), and Alexa Fluor 488 AffiniPure Donkey Anti-Chicken IgY (Jackson ImmunoResearch, 703-545-155).
Primary antibodies used for immunoblotting are the following: mouse anti-mNeongreen (1:1000; Chromotek, 32F6), rabbit anti-mNeongreen (1:1000; Cell Signaling Technology, 53061), mouse anti–α-tubulin (1:4000; Sigma-Aldrich, T6199), mouse anti-RFP (1:1000; Chromotek, 6G6), rabbit anti-GST (1:2000; Cell Signaling Technology, 2622), rabbit anti–phospho-AMPK (1:1000; Cell Signaling Technology, 2535), and mouse anti-AMPK (1:3000; Bio-Rad, MCA2672GA).
Secondary antibodies used in this study for immunoblotting are the following: horseradish peroxidase (HRP) goat anti-rabbit IgG (1:10,000; Abcam, ab6721) and HRP rabbit anti-mouse IgG (1:10,000; Abcam, ab6728).

For Co-IP assays, HEK-293T cells were transfected as indicated: 36 h later, cells were lysed in IP buffer (1% Triton X-100, 150 mM NaCl, and 50 mM Tris/HCl (pH 7.5), 1 mM PMSF, and protease inhibitor cocktail) and incubated with anti-Flag M2 affinity gels for 4 h at 4 °C. For reverse IP, HEK-293T cells were lysed and incubated with anti-HA antibody and protein A/G agarose for 12 h at 4 °C. Agarose was washed five times with IP assay buffer, boiled in SDS sample buffer, and subjected to Western blot analysis. Western blotting and immunofluorescence (IF) were performed as previously [40 (link)].
The following antibodies were used: mouse anti-Flag (F3165, Sigma-Aldrich), rabbit anti-Flag (F7425, Sigma-Aldrich), rabbit anti-GST (2622, Cell Signaling Technology), mouse anti-β-actin (66009-1-Ig, Proteintech), rabbit anti-Snail (3879, Cell Signaling Technology), mouse anti-Snail (sc-271977, Santa Cruz), rabbit anti-p66β (ab76925, Abcam), anti-biotin, HRP-linked (7075, Cell Signaling Technology), and rabbit anti-IgG (2729, Cell Signaling Technology), mouse anti-α-tubulin (66031-1-Ig, Proteintech), rabbit anti-Fibronectin1 (15613-1-AP, Proteintech), rabbit anti-E-cadherin (20874-1-AP, Proteintech), rabbit anti-Vimentin (10366-1-AP, Proteintech).

The following primary antibodies were used in this study: Rabbit anti-dsRed (Clontech #632496), Rat anti-E Cadherin (Life Technologies #13–1900), Rabbit anti-Fat3 and polyclonal Mouse anti-Fat3 (Lisa Goodrich, Harvard Medical School [18 (link)]), Goat anti-GFP (Abcam #5450, IF and WB), Goat anti-GFP (Abcam #6673, IP only), Rabbit anti-GST (Cell Signaling Technology #2625S) Mouse anti-HA (Covance #MMS-101P), Mouse anti-Kif1A (BD Biosciences #612094), Rabbit anti-Kif5B (Thermo # PA1-642), Goat anti-Kif5B (Imgenex #IMG-3049), Rabbit anti-Kif5C (Acris # SP5236P), Mouse anti-Phospho-Tyrosine (Cell Signaling Technology #9411). Fluorescent, Dylight conjugated secondary antibodies were purchased from Jackson Immunoresearch, and HRP conjugated secondary antibodies used for Western blot were from BioRad. Whole IgG negative controls for IP experiments were from Sino Biological (Goat IgG #CR2-500) and Millipore (Mouse IgG #12–371).

A standard Western blotting technique was performed. Briefly, the membranes were blocked using phosphate-buffered saline (PBS) containing 1% casein (Bio-Rad) and then probed with a 1:1,000 dilution of a primary antibody overnight at 4°C. The appropriate secondary antibody was used at a 1:10,000 dilution and incubated at room temperature for 1 h. Signal was detected with ECL+ (GE Healthcare), and the film was developed by a film processor (GE Healthcare). The antibodies used for Western blotting were mouse anti-Myc (Cell Signaling), rabbit anti-actin (Cell Signaling), rabbit anti-GST (Cell Signaling) antibodies and the custom-made EhAgo2-2 polyclonal antibodies in rabbit (28 (link)).

MEF, organoids and IEC samples were lysed in E1A buffer supplemented with sodium orthovanadate, sodium fluoride and cOmplete^™, EDTA-free Protease Inhibitor Cocktail (Roche) for 5–10 minutes on ice, spun down cold at maximum speed and protein concentration was measured with Bradford. 20–35 µg of protein was loaded on the gels and separated by SDS-PAGE (PAGE), transferred to nitrocellulose or PVDF (Millipore) membranes, and analyzed by immunoblotting. Proteins were detected with following antibodies: mouse anti-A20 (Santa Cruz sc-166692, 1:1000), rabbit anti-Atg16l1 (Cell Signaling 8089, 1:1000); rabbit anti-LC3 (MBL PM036, 1:1000); rabbit anti-p62 (MBL PM045, 1:2000), goat anti-IkBa (Santa Cruz sc-371-G, 1:1000); mouse anti-P-IkBa (Cell Signaling 9246, 1:1000); mouse anti-actin (MP Biomedicals 8691002, 1:10,000); mouse anti-HA (BioLegend 901501, 1:1000); mouse anti-Flag (Sigma F1804, 1:1000); rabbit anti-GST (Cell Signaling 2622, 1:1000); mouse anti-Tubulin (Sigma T4026, 1:40,000); mouse anti-GAPDH (Abcam Ab8245, 1:10,000); mouse anti-Atg16l1 (MBL 150-3, 1:2000); mouse anti-LC3 (MBL M186-3, 1:2000); mouse anti-UB (FK2; Millipore 4-263, 1:1000). As secondary antibodies, anti–rabbit, anti-mouse (GE Healthcare) or anti-goat (Santa Cruz)-HRP conjugates were used, and the signal was detected with enhanced chemiluminescence substrate ECL (Perkin Elmer).

Rabbit anti-FE65 and rabbit anti-APP A5137 were as described^{5 (link)}. Goat anti-FE65 E20 and mouse anti-α-tubulin DM1A were obtained from Santa Cruz. Mouse anti-myc 9B11, mouse anti-GFP JL8, mouse anti-HA 12CA5, rabbit anti-GST and mouse anti-APP 22C11 were purchased from Cell Signaling Technology, Clontech, Roche, Sigma and Millipore, respectively.
The phospho-specific antibody against phosphorylated FE65 at T579 (pT579 FE65) was generated by immunizing rats with a synthetic FE65 phosphopeptide (amino acids 575–586; REQW(pT)PSHVSVC) (GenScript) which contains a phosphorylated T579. A cysteine (C) residue was introduced to the peptide C-terminus for carrier protein conjugation and purification column coupling. Phosphopeptide conjugation was performed using the Imject Maleimide Activated mcKLH Spin kit (ThermoFisher Scientific). The immunized rat sera were purified by the non-phosphopeptide (REQWTPSHVSVC) and then phosphopeptide coupled columns prepared by using a SulfoLink Immobilization kit for peptides (ThermoFisher Scientific). The antibody was eluted and dialyzed against PBS/0.05% sodium azide, and then concentrated by using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane (Millipore).