Anti liprin alpha1

Primary antibodies used for this study were: anti-insulin (Dako Cytomation, A0564), anti-beta1 laminin (Thermo Scientific MA5-14657), anti-integrin beta 1 (BD Biosciences 555002), anti-talin (Sigma-Aldrich T3287), anti-phosphorylated FAK (Cell Signalling Tech 8,556S), anti-liprin alpha1 (Proteintech 14175-1-AP), and anti-ELKS (Sigma, E4531). All primary antibodies were diluted 1/200. Secondary antibodies were highly cross absorbed donkey or goat antibodies (Invitrogen) labelled with Alexa 488, Alexa 546, Alexa 594, or Alexa 647. All were used at a 1/200 dilution. DAPI (Sigma, 100 ng/ml final concentration) was added during the secondary antibody incubation.

Spheroids were fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 30 min at 20 °C. Samples were stored in PBS at 4 °C prior to immunofluorescent staining. Tissues were incubated in blocking buffer (3% BSA, 3% donkey serum, 0.3% Triton X-100) for a minimum of 40 min at room temperature followed by primary antibody incubation at 4 °C overnight. Sections were washed in PBS (4 changes over 30 min) and secondary antibodies (in block buffer) were added for 4 h at 20 °C. After washing in PBS, tissues were mounted in Prolong Diamond anti-fade reagent (Invitrogen). Primary antibodies used for this study were: anti-insulin (Dako Cytomation, A0564), anti-phosphorylated FAK (Cell Signalling Tech 8556S), anti-liprin alpha1 (Proteintech 14175-1-AP). All primary antibodies were diluted 1/200. Secondary antibodies were highly cross absorbed donkey or goat antibodies (Invitrogen) labelled with Alexa 488, Alexa 546, Alexa 594, or Alexa 647. All were used at a 1/200 dilution. DAPI (Sigma, 100 ng/mL final concentration) was added during the secondary antibody incubation. Confocal imaging was performed on a Leica SP8 microscope with a 63X oil immersion objective. The fluorescence intensity in regions of interest along the cell along the cell membrane was used to quantify the images.