Duolink in situ pla

PLAs were performed with Duolink in situ PLA (Sigma, mouse and rabbit probes) following the manufacturer’s instructions. A step by step protocol can be found in supplementary Methods, Supplementary Material online.

Keratinocytes from 1 or 2 day old C57Bl/6 newborn mouse skin were isolated as described (Wang et al., 2016 (link)), and cultured in FAD medium (low calcium, 0.07 mM) for 1 day. Calcium switch experiments (Wang et al., 2016 (link)) were performed by switching to FAD medium supplemented with with 1 mM CaCl₂. Keratinocytes were harvested for analysis at 4 days or at 36 hr after calcium switch as indicated in figure legends. For immunofluorescence, keratinocytes were fixed in 4% paraformaldehyde (PFA), blocked in 10% normal goat serum/0.1% Triton X-100/PBS for 1 hr at room temperature, incubated in primary antibody solution for 1 hr, washed in PBS, incubated in Alexa Fluor–conjugated secondary antibodies (Thermo Fisher Scientific), counterstained in DAPI (D1306, Thermo Fisher Scientific), and mounted in FluorSave Reagent mounting medium (345789, Calbiochem). Proximity ligation assay was performed according to the manufacturer’s protocol (Duolink in Situ PLA, Sigma-Aldrich). F-actin was stained using the Alexa Fluor 488 Phalloidin (A123791, Thermo Fisher Scientific) according to the manufacturer’s protocol. Micrographs were acquired using the Zeiss LSM 800 confocal microscope (Carl Zeiss Microscopy). Representative images from at least three independent experiments were shown. All images were and quantified by ImageJ software (NIH).

To detect protein–protein interaction, Duolink^® in situ PLA (Sigma Aldrich, MO, USA) was performed according to the manufacturer’s recommendations. HPF grown on chamber slide were fixed in 4% PFA for 10 min, permeabilized and blocked with 0.1% Tween 20 in 2% bovine serum albumin (BSA) for 1 h, and then incubated with primary antibodies, rabbit anti-TXNDC5 (1:1500, Proteintech, IL, USA, 19834-1-AP) together with mouse anti-TGFBR1 (1:100, Abnova, Taipei, Taiwan, H00007046-A01) or mouse anti-TGFBR2 (1:100, Abcam, Cambridge, UK, ab78419) overnight at 4 °C. After washing, slides were incubated with PLA probe for 1 h at 37 °C, followed by ligation and amplification. The fluorescence image was captured by fluorescence microscope. Slides without primary antibodies were used as negative control.

Duolink in situ PLA (DUO92014, Sigma-Aldrich) was performed in esophageal cancer cells. The paired-primary antibodies used in the present study were either rabbit anti-LOXL2, L2Δ13 or GAPDH antibody with mouse anti-aldolase A antibody. As a negative control, PLA was performed using only anti-LOXL2, anti-L2Δ13 or anti-aldolase A antibody, respectively. Briefly, cells were fixed with 4% paraformaldehyde for 10 min, washed three times with PBS, and permeabilized in 0.1% Triton X-100 for 10 min, followed by incubation with the indicated antibody pairs overnight at 4 °C. PLA was performed according to the manufacturer's recommendations. PLA imaging was conducted on a laser-scanning confocal microscope (LSM 800, Carl Zeiss) with a 63 × , 1.40 NA, Plan-apochromatic oil objective.