Anti ha antibody

NIH/3T3-Ago1, NIH/3T3-Ago2, or NIH/3T3-eGFP cells were cultured to ~70-80% confluency in 10-cm plates. Cells were fixed with 1% formaldehyde in serum-free media for 10 min at room temperature. Formaldehyde crosslinking was quenched with 125 µM glycine (final concentration). Cells were washed in PBS and nuclei were prepared as previously described (28 (link)). Cell suspension was sonicated 12 times in 10-s pulses to yield DNA fragments with an average length of ~500 bp. Lysates were pre-cleared with Protein A/G sepharose beads (GE Healthcare) and subsequently incubated with or without 5 µl of anti-HA antibody (Cell Signaling) at 4°C overnight. Samples omitting antibody were used as negative controls. Fresh beads were then added for 2 h to immunoprecipitate chromatin and sequentially washed in low salt, high salt, LiCl and TE buffer. DNA/protein complexes were eluted and crosslinking was reversed by heating overnight at 65°C. Samples were subsequently treated with Proteinase K followed by phenol/chloroform extraction and RNase A treatment. DNA fragments were then purified in nuclease free water using Qiaquick PCR purification columns (Qiagen). For RNAP II and H3K4me3 ChIP experiments, NIH/3T3 cells were transfected at 100 nM dsControl or miR-744 for 3 days, while TRAMP-C1-miR-EV and TRAMP-C1-miR-744 cells were cultured to ~70–80% confluency in 10 cm plates prior to harvest. Antibodies to RNAP II and H3K4me3 were purchased from Millipore. For ChIP–immunoblot analysis, NIH/3T3-Ago1 cells were transfected with 100 nM biotin-labeled miR-744 or dsControl for 3 days. Immunoprecipitation was performed with Dynabeads® M-280 Streptavidin (invitrogen) followed by immunoblotting with an anti-HA antibody.

HEK293T cells transfected to express the β-tubulin isotypes I, III, and V were grown in cell culture plates until sub-confluency and harvested. A cell pellet was prepared by centrifugation, washed twice with phosphate-buffered saline (PBS, Mediatech, Manassas, VA, USA), resolved in fixating solution consisting of 50% ethanol and 50% of PBS (10%)-buffered formalin, and fixed for a minimum of 6 h. After fixation, the pellet was collected by centrifugation, processed, and embedded in paraffin. The resulting blocks were sectioned at 5 μm thickness and mounted on Plus GOLD slides (Fisher Scientific, Schwerte, Germany).
Sections of formalin-fixed paraffin-embedded blocks of NSCLC tumour samples and transgenic control cell pellets were deparaffinised in xylene, dehydrated in a graded ethanol series, and incubated with blocking solution (Peroxid Block, Zytomed Systems, Berlin, Germany). Antigens were retrieved by heating samples in sodium citrate buffer, pH 6.0, for 20 min, and the following primary antibodies were used (Supplementary Figure 2): anti-HA antibody (mouse mAb, clone 6E2, Cell Signaling Technology, Danvers, MA, USA), anti-βIII-tubulin (mouse mAb, clone TU-20, Zytomed Systems), and anti-βV-tubulin (mouse mAb, clone 87CT59.3.7, Abgent, San Diego, CA, USA). Specific staining was detected using horseradish peroxidase (HRP)-labelled rabbit anti-mouse antibody (ZytoChemPlus (HRP) Polymer Kit, Zytomed Systems) and a 3,3′-diaminbenzidine (DAB) substrate system (DAB Substrate Kit, Zytomed Systems). Slides were counterstained with hematoxylin. Negative controls were performed by omitting the primary antibody. Furthermore, we used a brain metastasis containing a regular cortex with both neurons and glia cells, which was resected from a patient with a squamous cell carcinoma of the lung, for another set of positive and negative controls (Supplementary Figures 3A–C). Non-small cell lung cancer resection specimens having pleural nerves or nerve fascicles served as internal positive controls (Supplementary Figures 3D–F).
Immunohistochemistry evaluations were performed independently by one pathologist (JW) and one trained reader (DCC) who were blinded to the clinical data. Cytoplasmic immunoreactivity levels in each section were assessed under a light microscope and images were captured at 400-fold magnification. Tumour staining intensity was graded on a scale from 0+ to 3+ and each intensity category was scored a percentage of tumour cells ranging from 0 to 100 so that the sum of the percentages adds up to 100. The percentage score was then multiplied by its intensity category to obtain a final ‘H-score’, ranging from 0 to 300. Ambiguous cases were reevaluated jointly until a consensus was reached.