Tcs sp2

Coverslips carrying cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized with PBST (PBS containing 0.5% Triton X-100), and then blocked with 4% BSA (dissolved in PBST) overnight and incubated with anti-FLAG or anti-SBP antibodies. Secondary antibodies were FITC/TRITC-anti-mouse or anti-rabbit IgG. To visualize the preS that was bound on or internalized into the cells, as well as to observe the inhibition of preS internalization by anti-SBP pAb, cells in 24-well plates were pre-treated with or without anti-SBP antibody at 37°C for 30 min and then incubated with FITC-preS_1−131aa at 37°C for 60 min. Immunofluorescence assays were performed with Leica TCS SP2 and Olympus FV500 Confocal Laser Scanning microscopes.

For Van-FL labeling and detection, filaments were treated as described [10 (link)] and visualized with a Leica DM6000B fluorescence microscope and a FITCL5 filter (excitation band-pass, 480/40; emission band-pass, 527/30), and photographed with an ORCA-ER camera (Hamamatsu). GFP fluorescence was monitored with an Olympus TCS SP2 confocal laser-scanning microscope equipped with an HCX PLAN-APO 63 × 1.4 NA oil immersion objective (excitation, 488-nm; collection, 500–540 nm for GFP or 630–700 nm for cyanobacterial autofluorescence) or with an Olympus FLUOVIEW FV3000 (hyper-resolution) confocal laser-scanning microscope equipped with a UPlanApo 60 × 1.5 NA oil immersion objective (excitation, 488-nm; collection 500–540 nm for GFP or excitation, 640 nm; collection, 650–750 nm for cyanobacterial autofluorescence).

In Arabidopsis, whole-mount immunolocalization was performed following the published protocol^{69 (link)}. Antibodies were diluted as follows: 1:1000 for rabbit anti-PIN1^{15 (link)} (produced and processed in lab); 1:1000 for rabbit anti-PIN2^{70 (link)} (produced and processed in lab); and 1:600 for CY3-conjugated anti-rabbit secondary antibody (Sigma, C2306). In pea, water lanolin pastes containing IAA (0.16 μM), or IAA/GR24 (0.16 µM/0.09 µM) were applied on the stem stump or on the stem 2 mm below lateral incision. Immunolocalization was performed on longitudinal pea stem segments as described for Arabidopsis stem^{69 (link)}. The Arabidopsis anti-PIN1 antibody can also recognize the homologous PIN protein in pea^{4 (link)}. Antibodies were diluted as follows: 1:1000 for rabbit anti-PIN1^{15 (link)} (produced and processed in lab); and 1:500 for CY3-conjugated anti-rabbit secondary antibody (Sigma, C2306). All the fluorescence signals were evaluated on Zeiss LSM 700, Zeiss LSM 710, Zeiss Observer. Z1, Leica TCS SP2, Olympus Fluoview FV1000, or Olympus Fluoview 200 confocal scanning microscopes. Unless otherwise noted, the same microscope settings were usually used for each independent experiment and pixel intensities were taken into account when comparing the images between different samples. Images were finally assembled in Adobe Photoshop CC 2015 and Adobe Illustrator CS6.

Cells were grown on poly-l-lysine-coated sterile glass slide. After medium removal, cells were washed twice in PBS, thereafter fixed in cold Methanol for 5 min, then permeabilized for 10 min with 0.3% Triton X-100. Next, cells were rinsed three times with PBS and nonspecific binding sites were blocked with PBS 2% NGS (Normal Goat serum) for 30 min. Appropriate primary antibodies were dissolved in PBS 2% NGS and added for over night incubation. We used mouse anti-RTN-1C (1:100; Abcam, ab8961), rabbit anti-LC3 (1:200; Cell Signaling, 3868 S), mouse anti-LC3 (1:200; Nanotools, 0321-100/LC3-5F10), rabbit anti-ATG16L1 (1:200; Cell Signaling, 8089), rabbit anti-LAMP1 (1:100, MBL, ab 25630), mouse anti-HA (Boehringer, 1583816), rabbit anti-HA (Sigma, H6908) antibodies. After three washes in PBS, cells were incubated for 1 h with the appropriate anti-mouse and anti-rabbit secondary antibodies (Alexa, Molecular Probes, A11034, A11029, A11037) diluted 1:1000 in PBS 2% NGS. Finally, cell nuclei were stained with Hoechst 33342 (Molecular Probes). Slides were observed and photographed in a Leica TCS SP2 or Olympus IX81 (with FLUOVIEW 1000 confocal laser system) confocal microscope.