Alexa fluor 594 donkey anti rabbit antibody

Ipomoeassin F was chemically synthesized as described previously12 (link), 13 (link). PDI inhibitor CCF 642 (346640-08-2) was purchased from Tocris Bioscience (Shanghai, China). Thioflavin T (Th T) and Cycloheximide (CHX) were obtained from Sigma-Aldrich (USA). mRFP-GFP-MAP1LC3B plasmid was used as previously described14 (link). All antibody information is indicated as following: PDIA6 (#18233-1-AP, Proteintech), Calnexin (#10427-2-AP, Proteintech), GRP78/Bip (#11587-1-AP, Proteintech), ERp72 (PDIA4) (#2798, CST), eIF2α (#5324, CST), pho-eIF2α (#3597, CST), cleaved-caspase3 (#9664, CST), LC3B (#2775, CST), GAPDH (sc-3650620, Santa Cruz), α-Tubulin (sc-5286, Santa Cruz), anti-mouse linked with HRP (sc-2005, Santa Cruz), anti-rabbit IgG linked with HRP (sc-2004, Santa Cruz), Alexa Fluor 488 donkey anti-mouse antibody (#A21202, Thermo Fisher), and Alexa Fluor 594 donkey anti-rabbit antibody (#A21207, Thermo Fisher). Details for the use of antibodies can be found in Supplementary Table S2.

Flies were immobilized on ice and guts were dissected in ice-cold PBS. Dissected guts were immediately fixed in 4% formaldehyde for 30 min, washed in 0.2% Triton-X/PBS (PBST) and blocked in 5% BSA/PBS for 1 h on a shaker. Gut tissues were incubated with primary pH3 (CST, 9701; 1:500 dilution), dpErk (CST, 4370; 1:400 dilution) or lysozyme (Thermo Fisher Scientific, PA5-16668; 1:100 dilution) solutions in 5% BSA overnight at 4 °C, followed by incubation in secondary Alexa Fluor 594 donkey anti-rabbit antibody (Thermo Fisher Scientific, A21207; 1:1,000 dilution). Guts were mounted in mounting medium containing DAPI (Vectashield, H1200), scored and imaged using a Leica inverted microscope for the cell division assay and confocal SP8-DLS for the dpErk staining, with Leica Application Suite X software (v3.x, Leica Microsystems). pH3 and dpErk imaging was performed on the R2 region proximal to the proventriculus and for each intestine three adjacent images were taken.

hiPSCs were characterized quantitatively for pluripotency markers OCT4 and SSEA4 by flow cytometry. Briefly, the cells were detached using Stempro Accutase for 5 min at 37°C and washed with standard HuES media. The cells were then washed once with PBS and fixed with 4% PFA solution at a cell density of 1×10⁶ cells per ml at room temperature for 15 min. The cells were then permeabilized with 0.1% v/v Triton X-100 in HBSS for 15 min at room temperature, washed once with HBSS and incubated with 1% BSA in HBSS for 30 min. Following this, the cells were incubated with primary antibodies in 1% BSA in combination at the following dilutions, Rabbit Anti-OCT4 (Thermo Fisher Scientific, #A24867) at 1:100 and Mouse IgG3 Anti-SSEA4 (Thermo Fisher Scientific, #A24866) at 1:100 and incubated for 3 h at room temperature with gentle mixing. The cells were then washed two to three times in HBSS and incubated with appropriate secondary antibodies, Alexa Fluor™ 594 donkey anti-rabbit Antibody (Thermo Fisher Scientific, #A24870) and Alexa Fluor™ 488 goat anti-mouse IgG3 (Thermo Fisher Scientific, #A24877) for 1 h at room temperature. Before use, the secondary antibodies were diluted at 1:250 in 1% BSA. The cells were washed twice and then analyzed using a BD FACSVerse cytometer (Fig. S1A–C).