*2.6. Western Blotting*

Generation of whole cell lysates was carried out by scraping cells off into ice-cold RIPA buffer (150 mmol/L NaCl, 1% NP40, 0.5% sodium-deoxycholate, 0.1% sodium-dodecylsulfate, 50 mmol/L Tris/HCl, pH 8, 10 mmol/L NaF, 1 mmol/L Na3VO4) supplemented with Protease-Inhibitor cocktail (Roche). After 2–3 freeze and thaw cycles the protein content of the lysates was measured by using DC™ Protein Assay (Bio-Rad). 50 μg to 100 μg of protein were loaded onto SDS-PAGE electrophoresis. Western blots were done as previously described [11,21] and the indicated antibodies were used to detect protein expression.

### *2.7. Real-Time Reverse Transcription PCR (qRT-PCR)*

RNA was isolated using RNeasy Mini Kit (74106, Qiagen, Hilden, Germany) according to the manufacturer's instruction and as previously described [11,22]. Expression levels were normalized to the reference gene (β-actin; set as 1) and were shown as relative quantification. Specific primers were designed using Primer 3 [23] based on available NCBI nucleotide CDS sequences. Cross-reaction of primers was excluded by comparison of the sequence of interest with the NCBI database (Blast 2.2, U.S. National Centre for Biotechnology Information, Bethesda, MD) and all primers used were intron-spanning. PCR products are 200-300 bp in size. qRT-PCR was carried out using specific oligonucleotide primers (s sense, as antisense; TRIAP1s AGGATTTCGCAAGTCCAGAA, TRIAP1as GCTGATTCCACCCAAGTAT; TAGLNs TCCAGACTGTTGACCTCTTTGA, TAGLNas CCTCTCCGC TCTAACTGATGAT; ACTA2s GCCGAGATCTCACTGACTACCT, ACTA2as TGATGCTGTTGTAGGTG GTTTC; TGFB1s CCCACAACGAAATCTATGACAA, TGFB1as AACTCCGGTGACATCAAAAGAT; LAMP1s CCTGCCTTTAAAGCTGCCAA; LAMP1as CACCTTCCACCTTGAAAGCC; LAMP2s ACC ACTGTGCCATCTCCTAC, LAMP2as TGCCTGTGGAGTGAGTTGTA; ACTINs GGCACCACACTTT CTACAATGA, ACTINas TCTCTTTAATGTCACGCACGAT) as previously described [11,22].
