Mouse anti kdel

The chitin-binding probe was prepared in our laboratory as described [91 (link)]. Chitin-546 was used at 1:10 dilution. The following primary antibodies were used: Chicken anti-GFP (1:200) (Abcam), Guinea Pig anti-Uif (1:300) [69 (link)], Mouse anti-Gasp (1:5) (DSHB), Guinea Pig anti-Cor (1:500) [66 (link)], Rabbit anti-Verm (1:100) [27 (link)], Rabbit anti-Kune-kune (1:200) [43 (link)], Mouse anti-DLG (1:1) (DSHB), Mouse anti-Spectrin (1:10) (DSHB), Mouse anti-Cnx99A (1:5) (DSHB), Rabbit anti-GM130 (1:200) (Abcam), Mouse anti-KDEL (1:200) (Abcam), Rabbit anti-Rab5 (1:200) (Abcam), Rat anti-Rab11 (1:100) [92 (link)], and Mouse anti-Rab7 (1:10) (DSHB). Antibodies generated in this study: Rabbit anti-Osi19 (1:300), Rat anti-Osi15 (1:25), Guinea Pig anti-Osi9 (1:10).
The following secondary antibodies (Invitrogen life technologies) were used: AlexaFluor 488 anti-chicken IgG, anti-rat IgG, anti-mouse IgG, anti-rabbit IgG, anti-guinea pig IgG; AlexaFluor 543 anti-rat IgG, anti-rabbit IgG, anti-guinea pig IgG, anti-mouse IgG; AlexaFluor 594/546 anti-mouse IgM; AlexaFluor 647 anti-rat IgG, anti-mouse IgG, anti-rabbit IgG, anti-guinea pig IgG; AlexaFluor 647 plus anti-mouse, anti-rabbit; AlexaFluor 488 plus anti-mouse, anti-rabbit), and AlexaFluor 405 anti-Guinea Pig. All secondary antibodies were used at 1:200 dilution.

Plasmid allowing the expression of PFO in E. coli was kindly offered by Daniel Portnoy (DP-4167) [42 (link)]. Plasmid GFPNMHCIIA was a gift from Robert Adelstein through Addgene (#11347) [43 (link)], plasmid tdTomato-F-Tractin [44 (link)] was kindly offered by John Hammer (NIH, Bethesda, MD, USA), and plasmid pUBC eGFP-KDEL was a gift from François-Xavier Campbell-Valois [45 (link)]. The following antibodies were used at 1/200 for immunofluorescence microscopy (IF): rabbit anti-NMHCIIA (Sigma, St Louis, MO, USA), mouse anti-NMHCIIA (Abcam, Cambridge, UK), and mouse anti-KDEL (Abcam, Cambridge, UK). Secondary antibodies were used at 1/200: goat anti-rabbit Alexa Fluor 488 (Invitrogen, Waltham, MA, USA) and goat anti-mouse Cy3 (Jackson ImmunoResearch, West Grove, PA, USA). For IF, F-actin was labeled with rhodamine phalloidin (Invitrogen, Waltham, MA, USA) and the PM with FITC-conjugated WGA (Sigma, St Louis, MO, USA) that recognizes sialic acid and N-acetylglucosaminyl sugar residues at the PM.

Immunostainings were performed using standard protocols. The following primary antibodies were used: rabbit anti-SKTL at 1:20000 (Claret et al., 2014 (link)); mouse anti-Myc 9E10 (Santa Cruz) at 1:250; rabbit anti-RAB11 at 1:8000 (Nakamura); rabbit anti-RAB5 at 1:50 (Marcos Gonzalez-Gaitan); guinea pig anti-HRS at 1:500 (H. Bellen); mouse anti-KDEL at 1:300 (abcam 10C3, RRID: AB_298945); rabbit anti-NUF at 1:500; mouse anti-spNF at 1:50 (Abdu Uri); rabbit anti-Syntaxin16 at 1:1000 (R.Leborgne); rabbit anti-PKC (Santa Cruz) at 1:1000; mouse anti-HTS (Hybridoma, RRID: AB_528289) at 1:10; mouse anti-ß-Gal (Promega) at 1:250; rabbit anti-Staufen at 1:200. F-actin was visualised after staining with rhodamine-phalloidin (RRID: AB_2572408) or Alexa488-phalloidin (RRID: AB_2315147; life technology) at 1:200. Alexa594-WGA (Molecular probes) was used at 1:100 to stain nuclear membrane. Images were obtained with a ZEISS LSM700 confocal microscope and a Leica TCS-SP5 AOBS inverted scanning microscope.